Compositions and Methods for Detecting Microorganisms

ABSTRACT

Described herein are compositions and methods for detecting the presence or absence of a microorganism in a sample comprising contacting the sample with an aptamer capable of binding to a cell-surface protein of the microorganism to form a complex, contacting the mixture with a second aptamer capable of binding to the first cell-surface protein or a second cell-surface protein of the microorganism; and performing an assay to detect the second aptamer, wherein detecting the second aptamer indicates that the microorganism is present in the sample, and wherein not detecting the second aptamer indicates that the microorganism is absent from the sample.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 61/940,955, filed Feb. 18, 2014, and U.S. Provisional ApplicationSer. No. 61/947,627, filed Mar. 4, 2014, each of which is incorporatedherein by reference in its entirety.

FIELD

The present disclosure relates generally to composition and methods fordetecting for the presence of a microorganism in a sample. Morespecifically, the disclosure relates to nucleic acid aptamers capable ofbinding microorganism protein, and methods for the capture and detectionof a microorganism with nucleic acid aptamers in a sample.

Incorporated by reference herein in its entirety is the Sequence Listingentitled “Sequence_Listing_ST25”, created Feb. 13, 2015 size of 16kilobytes.

BACKGROUND

The contamination of food and water poses a major health risk in bothdeveloped countries and third world countries, and is thought to beresponsible for millions of human deaths and illnesses annually.Moreover, contamination to food and water also threatens animal health,including livestock and aquatic ecosystems.

Generally, these illnesses are caused by microorganism contamination,such as bacteria, parasites or viruses. With respect to food production,the complexity and the number of parties involved provide an abundantnumber of opportunities for unintentional contamination, and thepotential and unfortunate interplay of terrorism and food supply.Surface and ground water generally become contaminated by pets,livestock or wild animal defecating in or near a water source, whilerun-off from landfills, septic fields, sewers and agricultural landsalso contribute to water contamination. Irrespective of the type andsource of contamination, it can be difficult for individuals todetermine if food or water is contaminated because it may appear andtaste fine, but still cause illness and ultimately death. Thus,monitoring for microbial contamination of food, water, non-sterileproducts, or the environment is critical to public health on a globalscale

Therefore, there continues to be a need for alternative compositions andmethods for improved, cost-effective and efficient monitoring formicrobial contamination in both food and water. The present disclosuremeets such needs by providing novel aptamer reagents with highspecificity and affinity for cell surface epitopes on a microorganismfor the capture and enrichment of a microorganism present at low celldensities and for the direct detection (e.g., by qPCR or fluorescentstaining) without the need for culture or cell lysis.

SUMMARY

The present disclosure describes the generation of novel slow off-ratemodified aptamer (SOMAmer) reagents to several Staphylococcus aureuscell surface-associated proteins via SELEX with multiple modified DNAlibraries using purified recombinant or native proteins. High-affinitybinding agents with sub-nanomolar K_(d)'s were obtained forstaphylococcal protein A (SpA), clumping factors (ClfA, ClfB),fibronectin-binding proteins (FnbA, FnbB) and iron-regulated surfacedeterminants (Isd). Several aptamers specifically bound to S. aureuscells from all strains that were tested, but not to other staphylococcior other bacteria. SpA and ClfA aptamers proved useful for the selectivecapture and enrichment of S. aureus cells from low cell-densitymatrices, as shown by culture and PCR, leading to improved limits ofdetection and efficient removal of PCR inhibitors. Detection of S.aureus cells was enhanced by several orders of magnitude when thebacterial cell surface was coated with aptamers followed by qPCR of theaptamers compared to genomic PCR.

The present disclosure describes a method for detecting the presence orabsence of a microorganism in a sample comprising: a) contacting thesample with a first aptamer to form a mixture, wherein the first aptameris capable of binding to a first cell-surface protein of themicroorganism to form a complex and comprises a first tag, wherein thefirst tag is capable of binding to a solid support; b) contacting themixture with the solid support under conditions that permit the firsttag to bind to the solid support; c) washing the solid support to enrichthe mixture for the complex and/or washing the solid support tosubstantially remove unbound material; c) contacting the mixture with asecond aptamer, wherein the second aptamer is capable of binding to thefirst cell-surface protein or a second cell-surface protein of themicroorganism; and d) performing an assay to detect the second aptamer,wherein detecting the second aptamer indicates that the microorganism ispresent in the sample, and wherein not detecting the second aptamerindicates that the microorganism is absent from the sample.

The present disclosure further provides for a method for detecting thepresence or absence of a microorganism in a sample comprising:

a) contacting the sample with a solid support, wherein a first aptameris bound to the solid support via a first tag, and wherein the firstaptamer is capable of binding to a first cell-surface protein of themicroorganism to form a complex; b) washing the solid support to enrichthe mixture for the complex and/or washing the solid support tosubstantially remove unbound material; c) contacting the mixture with asecond aptamer, wherein the second aptamer is capable of binding to thefirst cell-surface protein or a second cell-surface protein of themicroorganism and comprises a second tag; and d) performing an assay todetect the second aptamer, wherein detecting the second aptamerindicates that the microorganism is present in the sample, and whereinnot detecting the second aptamer indicates that the microorganism isabsent from the sample.

In another aspect, the at least one of the first aptamer and the secondaptamer further comprise at least one C-5 modified pyrimidine. In arelated aspect, the C-5 modified pyrimidine is selected from the groupconsisting of 5-(N-benzylcarboxyamide)-2′-deoxycytidine (BndC);5-(N-2-phenylethylcarboxyamide)-2′-deoxycytidine (PEdC);5-(N-3-phenylpropylcarboxyamide)-2′-deoxycytidine (PPdC);5-(N-1-naphthylmethylcarboxyamide)-2′-deoxycytidine (NapdC);5-(N-2-naphthylmethylcarboxyamide)-2′-deoxycytidine (2NapdC);5-(N-1-naphthyl-2-ethylcarboxyamide)-2′-deoxycytidine (NEdC);5-(N-2-naphthyl-2-ethylcarboxyamide)-2′-deoxycytidine (2NEdC);5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU);5-(N-isobutylcarboxyamide)-2′-deoxyuridine (iBudU);5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU);5-(N-[1-(3-trimethylammonium) propyl]carboxyamide)-2′-deoxyuridinechloride and 5-(N-naphthylmethylcarboxyamide)-2′-deoxyuridine (NapdU).

In another aspect, the second aptamer is amplifiable. In a relatedaspect, the second aptamer is a template for enzymatic amplifications(e.g. by PCR or qPCR). In yet another related aspect, the second aptameris amplified by PCR primers that are capable of hybridizing with thesecond aptamer or one or more regions of the second aptamer.

In another aspect, the second aptamer comprises a second tag, whereinthe second tag is selected from the group consisting of a dye, a quantumdot, a radiolabel, PCR primer sites, an electrochemical functionalgroup, and an enzyme plus a detectable enzyme substrate.

In another aspect, the first tag is selected from the group consistingof a polynucleotide, a polypeptide, a peptide nucleic acid, a lockednucleic acid, an oligosaccharide, a polysaccharide, an antibody, anaffibody, an antibody mimic, a cell receptor, a ligand, a lipid, biotin,polyhistidine, or any fragment or derivative of these structures.

In another aspect, solid support is selected from the group consistingof a bead and a substrate. In a related aspect, the bead is selectedfrom the group consisting of a polymer bead, an agarose bead, apolystyrene bead, an acrylamide bead, a solid core bead, a porous bead,a paramagnetic bead, glass bead, microbead, and controlled pore bead. Inyet another related aspect, the substrate is selected from the groupconsisting of a microtiter well, a cyclo-olefin copolymer substrate, amembrane, a plastic substrate, nylon, a Langmuir-Blodgett film, glass, agermanium substrate, a silicon substrate, a silicon wafer chip, a flowthrough chip, a nanoparticle, a polytetrafluoroethylene substrate, apolystyrene substrate, a gallium arsenide substrate, a gold substrate,and a silver substrate.

In another aspect, the assay is selected from the group including butnot limited to PCR, qPCR, mass spectroscopy, sequencing hybridizationand the like. In a related aspect, the assay is selected from the groupconsisting of PCR and qPCR.

In another aspect, the microorganism is selected from the groupincluding, but not limited to a bacterial cell, parasite and virus.

In another aspect, the microorganism is a bacterial cell. In a relatedaspect, the bacterial cell is pathogenic. In yet another related aspect,the bacterial cell is a Staphylococcus cell. In another related aspect,the bacterial cell is a Staphylococcus aureus cell.

In another aspect, the first cell-surface protein and the secondcell-surface protein are the same protein or a different protein.

In another aspect, the first cell-surface protein is a bacterialcell-surface protein.

In another aspect, the second cell-surface protein is a bacterialcell-surface protein.

In another aspect, the first cell-surface protein is selected from thegroup consisting of SPA, ClfA, ClfB, FnbA, FnbB, IsdA, IsdB, IsdC, IsdHand SasD. In a related aspect, the first cell-surface protein isselected from the group consisting of SPA and ClfA.

In another aspect, the second cell-surface protein is selected from thegroup consisting of SPA, ClfA, ClfB, FnbA, FnbB, IsdA, IsdB, IsdC, IsdHand SasD. In a related aspect, the second cell-surface protein isselected from the group consisting of SPA and ClfA.

In another aspect, the first aptamer comprises a nucleic acid moleculehaving the sequence of GGCWWCGGGWACCWAWWAWNGGWWWAGCC(N)_(n)GWC (SEQ IDNO: 14), wherein W is independently, for each occurrence, a C-5 modifiedpyrimidine, N is any unmodified or modified nucleotide, and n is 0, 1,2, 3, 4 or 5. In a related aspect, n is 2. In a related aspect, thefirst aptamer is at least about 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides in length. In yetanother related aspect, the first aptamer is at from about 32 to about100 nucleotides in length (or 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99 or 100 nucleotides in length).

In another aspect, the first aptamer comprises a nucleic acid moleculehaving the sequence of AWCWGGWWC(N)_(n)AWCWGGWWWWWAAG (SEQ ID NO: 15),wherein W is independently, for each occurrence, a C-5 modifiedpyrimidine, N is any unmodified or modified nucleotide, and n is 0, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29 or 30. In a related aspect, n is from 5to 20 (or 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20).In another aspect, n is from 10 to 18 (or 10, 11, 12, 13, 14, 15, 16, 17or 18). In a related aspect, n is about 16. In yet another relatedaspect, the first aptamer is at least about 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49 or 50 nucleotides in length.

In another aspect, the first aptamer is from about 18 to about 100nucleotides in length (or 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or100 nucleotides in length).

In another aspect, the first aptamer comprises a nucleic acid moleculehaving a sequence selected from the group consisting of SEQ ID NOs: 1-8and 10-12, wherein W is a C-5 modified pyrimidine.

In another aspect, the second aptamer comprises a nucleic acid moleculehaving the sequence of GGCWWCGGGWACCWAWWAWNGGWWWAGCC(N)_(n)GWC (SEQ IDNO:14), wherein W is independently, for each occurrence, a C-5 modifiedpyrimidine, N is any unmodified or modified nucleotide, and n is 0, 1,2, 3, 4, or 5. In a related aspect, n is 2. In a related aspect, thesecond aptamer is at least about 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides in length. In yetanother related aspect, the second aptamer is at from about 32 to about100 nucleotides in length (or 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99 or 100 nucleotides in length).

In another aspect, the second aptamer comprises a nucleic acid moleculehaving the sequence of AWCWGGWWC(N)_(n)AWCWGGWWWWWAAG (SEQ ID NO:15),wherein W is independently, for each occurrence, a C-5 modifiedpyrimidine, N is any unmodified or modified nucleotide, and n is 0, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29 or 30. In a related aspect, n is from 5to 20 (or 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20).In another aspect, n is from 10 to 18 (or 10, 11, 12, 13, 14, 15, 16, 17or 18). In a related aspect, n is about 16.

In another aspect, the second aptamer is at least about 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides in length.

In another aspect, the second aptamer is from about 18 to about 100nucleotides in length (or 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or100 nucleotides in length).

In another aspect, the second aptamer comprises a nucleic acid moleculehaving a sequence selected from the group consisting of SEQ ID NOs: 1-8and 10-12, wherein W is a C-5 modified pyrimidine.

In another aspect, the C-5 modified pyrimidine is selected from thegroup consisting of 5-(N-benzylcarboxyamide)-2′-deoxycytidine (BndC);5-(N-2-phenylethylcarboxyamide)-2′-deoxycytidine (PEdC);5-(N-3-phenylpropylcarboxyamide)-2′-deoxycytidine (PPdC);5-(N-1-naphthylmethylcarboxyamide)-2′-deoxycytidine (NapdC);5-(N-2-naphthylmethylcarboxyamide)-2′-deoxycytidine (2NapdC);5-(N-1-naphthyl-2-ethylcarboxyamide)-2′-deoxycytidine (NEdC);5-(N-2-naphthyl-2-ethylcarboxyamide)-2′-deoxycytidine (2NEdC);5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU);5-(N-isobutylcarboxyamide)-2′-deoxyuridine (iBudU);5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU);5-(N-[1-(3-trimethylammonium) propyl]carboxyamide)-2′-deoxyuridinechloride and 5-(N-naphthylmethylcarboxyamide)-2′-deoxyuridine (NapdU).

In another aspect, the sample is selected from the group including, butnot limited to a water sample, a soil sample, a food sample, a cellsample, a culture sample, a tissue sample, a cell debris sample abiological sample and the like.

In another aspect, and for any of the embodiments disclosed herein, theconcentration of the first aptamer is from about 0.5 nmol 1⁻¹ to about60 nmol 1⁻¹ (or 0.5, 1, 1.5, 2, 2.5, 3, 3.2, 3.5, 4, 4.5, 5.5, 6, 6.5,7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14,14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21,21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28,28.5, 29, 29.5, 30, 30.5, 31, 31.5, 32, 32.5, 33, 33.5, 34, 34.5, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59 or 60 nmol 1⁻¹). In a related aspect, theconcentration of the first aptamer is from about 1 nmol 1⁻¹ to about 40nmol 1⁻¹ (or 1, 1.5, 2, 2.5, 3, 3.2, 3.5, 4, 4.5, 5.5, 6, 6.5, 7, 7.5,8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15,15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22,22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29,29.5, 30, 30.5, 31, 31.5, 32, 32.5, 33, 33.5, 34, 34.5, 35, 36, 37, 38,39 or 40 nmol 1⁻¹). In a related aspect, the concentration of the firstaptamer is from about 2 nmol 1⁻¹ to about 35 nmol 1⁻¹ (or, 2, 2.5, 3,3.2, 3.5, 4, 4.5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11,11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18,18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25,25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 30.5, 31, 31.5, 32,32.5, 33, 33.5, 34, 34.5 or 35 nmol 1⁻¹).

In a related aspect, the concentration of the first aptamer is at least0.5 nmol 1⁻¹, 1 nmol 1⁻¹, 2 nmol 1⁻¹, 3 nmol 1⁻¹, 3.2 nmol 1⁻¹, 4 nmol1⁻¹, 5 nmol 1⁻¹, 6 nmol 1⁻¹, 7 nmol 1⁻¹, 8 nmol 1⁻¹, 9 nmol 1⁻¹, 10 nmol1⁻¹, 11 nmol 1⁻¹, 12 nmol 1⁻¹, 13 nmol 1⁻¹, 14 nmol 1⁻¹, 15 nmol 1⁻¹, 16nmol 1⁻¹, 17 nmol 1⁻¹, 18 nmol 1⁻¹, 19 nmol 1⁻¹, 20 nmol 1⁻¹, 21 nmol1⁻¹, 22 nmol 1⁻¹, 23 nmol 1⁻¹, 24 nmol 1⁻¹, 25 nmol 1⁻¹, 26 nmol 1⁻¹, 27nmol 1⁻¹, 28 nmol 1⁻¹, 29 nmol 1⁻¹, 30 nmol 1⁻¹, 31 nmol 1⁻¹, 32 nmol1⁻¹, 33 nmol 1⁻¹, 34 nmol 1⁻¹, 35 nmol 1⁻¹, 36 nmol 1⁻¹, 37 nmol 1⁻¹, 38nmol 1⁻¹, 39 nmol 1⁻¹, or 40 nmol 1⁻¹. In a related aspect, theconcentration of the first aptamer is at least 1 nmol 1⁻¹. In a relatedaspect, the concentration of the first aptamer is at least 3 nmol 1⁻¹.In a related aspect, the concentration of the first aptamer is at least5 nmol 1⁻¹. In a related aspect, the concentration of the first aptameris at least 10 nmol 1⁻¹. In a related aspect, the concentration of thefirst aptamer is at least 20 nmol 1⁻¹. In a related aspect, theconcentration of the first aptamer is at least 30 nmol 1⁻¹. In a relatedaspect, the concentration of the first aptamer is at least 32 nmol 1⁻¹.

In another aspect, and for any of the embodiments disclosed herein, theconcentration of the second aptamer is from about 5 nM to about 200 nm(or 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145,150, 155, 160, 165, 170, 175, 180, 185, 190, 195 or 200 nM). In arelated aspect, the concentration of the second aptamer is from about2.5 nM to about 100 nM (or 2.5, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 nM). In a related aspect, theconcentration of the second aptamer is from about 10 nM to about 100 nM(or 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,95 or 100 nM).

In a related aspect, the concentration of the second aptamer is at least2.5 nM, 3 nM, 5 nM, 10 nM, 15 nM, 20 nM, 25 nM, 30 nM, 35 nM, 40 nM, 45nM, 50 nM, 55 nM, 60 nM, 65 nM, 70 nM, 75 nM, 80 nM, 85 nM, 90 nM, 95 nMor 100 nM. In a related aspect, the concentration of the second aptameris at least 2 nM. In a related aspect, the concentration of the secondaptamer is at least 2.5 nM. In a related aspect, the concentration ofthe second aptamer is at least 5 nM. In a related aspect, theconcentration of the second aptamer is at least 10 nM. In a relatedaspect, the concentration of the second aptamer is at least 20 nM. In arelated aspect, the concentration of the second aptamer is at least 30nM. In a related aspect, the concentration of the second aptamer is atleast 40 nM. In a related aspect, the concentration of the secondaptamer is at least 50 nM. In a related aspect, the concentration of thesecond aptamer is at least 60 nM. In a related aspect, the concentrationof the second aptamer is at least 70 nM. In a related aspect, theconcentration of the second aptamer is at least 80 nM. In a relatedaspect, the concentration of the second aptamer is at least 90 nM. In arelated aspect, the concentration of the second aptamer is at least 100nM.

The present disclosure further describes a nucleic acid moleculecomprising at least about 15 to at least about 100 nucleotides (or atleast about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100nucleotides), at least one C-5 modified pyrimidine, and is capable ofbinding to a cell-surface protein of a microorganism.

In another aspect, the nucleic acid molecule comprises from about 15 toabout 50 nucleotides (or 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49 or 50 nucleotides).

In another aspect, the nucleic acid molecule comprises at least 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 C-5modified pyrimidines.

In another aspect, the nucleic acid molecule comprises at least 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more C-5 modified pyrimidines.

In another aspect, the nucleic acid molecule is capable of binding tothe cell-surface protein with an equilibrium binding constant (K_(d)) offrom about 0.03 nM to about 4.7 nM (or 0.03, 0.04, 0.05, 0.06, 0.07,0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19,0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31,0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43,0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75,0.8, 0.85, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1,2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6,3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7 nM)

In another aspect, nucleic acid molecule is capable of binding to thecell-surface protein with an equilibrium binding constant (Kd) of atleast about 0.03, 0.07, 0.08, 0.14, 0.15, 0.16, 0.22, 0.35, 0.47, 0.63,0.73, 0.79, 0.84, 1.3, 1.35, 1.98, 2.17, 3.9 and 4.73 nM.

In another aspect, the C-5 modified pyrimidine is selected from thegroup consisting of 5-(N-benzylcarboxyamide)-2′-deoxycytidine (BndC);5-(N-2-phenylethylcarboxyamide)-2′-deoxycytidine (PEdC);5-(N-3-phenylpropylcarboxyamide)-2′-deoxycytidine (PPdC);5-(N-1-naphthylmethylcarboxyamide)-2′-deoxycytidine (NapdC);5-(N-2-naphthylmethylcarboxyamide)-2′-deoxycytidine (2NapdC);5-(N-1-naphthyl-2-ethylcarboxyamide)-2′-deoxycytidine (NEdC);5-(N-2-naphthyl-2-ethylcarboxyamide)-2′-deoxycytidine (2NEdC);5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU);5-(N-isobutylcarboxyamide)-2′-deoxyuridine (iBudU);5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU);5-(N-[1-(3-trimethylammonium) propyl]carboxyamide)-2′-deoxyuridinechloride and 5-(N-naphthylmethylcarboxyamide)-2′-deoxyuridine (NapdU).

In another aspect, the microorganism is selected from the groupconsisting of a bacterial cell, parasite and virus.

In another aspect, the microorganism is a bacterial cell. In a relatedaspect, the bacterial cell is pathogenic.

In another aspect, the bacterial cell is a Staphylococcus cell. In arelated aspect, the bacterial cell is a Staphylococcus aureus cell.

In another aspect, the cell-surface protein is a bacterial cell-surfaceprotein.

In another aspect, the cell-surface protein is selected from the groupconsisting of SPA, ClfA, ClfB, FnbA, FnbB, IsdA, IsdB, IsdC, IsdH andSasD.

In another aspect, the first cell-surface protein is selected from thegroup consisting of SPA and ClfA.

In another aspect, the nucleic acid molecule comprises the sequence ofGGCWWCGGGWACCWAWWAWNGGWWWAGCC(N)_(n)GWC (SEQ ID NO: 14), wherein W isindependently, for each occurrence, a C-5 modified pyrimidine, N is anyunmodified or modified nucleotide, and n is 0, 1, 2, 3, 4 or 5. In arelated aspect, n is 2.

In another aspect, the nucleic acid molecule is at least about 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50nucleotides in length.

In another aspect, the nucleic acid molecule is at from about 32 toabout 100 nucleotides in length (or 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99 or 100 nucleotides in length).

In another aspect, the nucleic acid molecule comprises the sequence ofAWCWGGWWC(N)_(n)AWCWGGWWWWWAAG (SEQ ID NO:15), wherein W isindependently, for each occurrence, a C-5 modified pyrimidine, N is anyunmodified or modified nucleotide, and n is 0, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29 or 30. In a related aspect, n is from 5 to 20 (or 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20). In another aspect, nis from 10 to 18 (or 10, 11, 12, 13, 14, 15, 16, 17 or 18). In a relatedaspect, n is about 16.

In another aspect, the nucleic acid molecule is at least about 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides inlength.

In another aspect, the nucleic acid molecule is from about 18 to about100 nucleotides in length (or 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99or 100 nucleotides in length).

In another aspect, the nucleic acid molecule comprises a nucleic acidmolecule having a sequence selected from the group consisting of SEQ IDNOs: 1-8 and 10-12, wherein W is a C-5 modified pyrimidine.

In another aspect, the C-5 modified pyrimidine is selected from thegroup consisting of 5-(N-benzylcarboxyamide)-2′-deoxycytidine (BndC);5-(N-2-phenylethylcarboxyamide)-2′-deoxycytidine (PEdC);5-(N-3-phenylpropylcarboxyamide)-2′-deoxycytidine (PPdC);5-(N-1-naphthylmethylcarboxyamide)-2′-deoxycytidine (NapdC);5-(N-2-naphthylmethylcarboxyamide)-2′-deoxycytidine (2NapdC);5-(N-1-naphthyl-2-ethylcarboxyamide)-2′-deoxycytidine (NEdC);5-(N-2-naphthyl-2-ethylcarboxyamide)-2′-deoxycytidine (2NEdC);5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU);5-(N-isobutylcarboxyamide)-2′-deoxyuridine (iBudU);5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU);5-(N-[1-(3-trimethylammonium) propyl]carboxyamide)-2′-deoxyuridinechloride and 5-(N-naphthylmethylcarboxyamide)-2′-deoxyuridine (NapdU).

The present disclosure further describes a kit for detecting thepresence or absence of a microorganism in a sample comprising a nucleicacid molecule as described above.

The foregoing and other objects, features, and advantages of theinvention will become more apparent from the following detaileddescription, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the capture of Staphylococcus aureus bacteria with SpAaptamers immobilized on paramagnetic beads. The efficiency of cellcapture was calculated via quantitative culture of the beads. In FIG. 1Athe aptamer concentration was fixed at 20 nmol 1⁻¹ to capture cells in a0.1 ml sample. (▪) 5000 CFU, (

) 500 CFU, and (

) 50 CFU. In FIG. 1B the cell density was fixed at 6600 CFU in a 0.1 mlsample and the capture aptamer concentrations were varied. (▪) 32 nmol1⁻¹, (

) 10 nmol 1⁻¹, (

) 3.2 nmol 1⁻¹, (

) 1 nmol 1⁻¹, and (

) 0.32 nmol 1⁻¹. The efficiency of capture was calculated viaquantitative culture.

FIG. 2 shows aptamer-based capture of Staphylococcus aureus, and signalamplification via qPCR of aptamers bound to highly abundant cell surfacecomponents compared to qPCR of single genomic copies. Non-amplifiable,biotinylated ClfA aptamers 4522-5 or 4503-73 were used for capture of S.aureus or S. epidermidis (negative control, followed by detection withamplifiable SpA aptamer 4520-8. Random aptamer library was used asnegative control for detection. PCR amplification of a genomic target(sasD gene) was performed for reference, using the same cell titer (10⁸cells ml⁻¹).

FIG. 3 shows an SDS-PAGE analysis of cell surface-associated S. aureusproteins over-expressed in recombinant form in E. coli and purified byaffinity chromatography on Ni-NTA agarose and Streptactin Sepharose.

FIG. 4 shows a radiolabel affinity binding assays with individualaptamers from SELEX pool 4520 NapdU and 4531 TrpdU using purified SpAprotein serially diluted from 0.001-100 nmol 1⁻¹ (FIG. 4A) and wholecells diluted to 10⁷, 10⁶, 10⁵, and 10⁴ CFU ml⁻¹ (FIG. 4B).

FIG. 5 shows the capture of bacterial cells with SpA and ClfA aptamersimmobilized on paramagnetic streptavidin beads. Efficiency of capturewas monitored by semi-quantitative culture at low cell density (FIG. 5A)or by decrease in turbidity at high cell density (FIG. 5B).

DETAILED DESCRIPTION I. Terms and Methods

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of thedisclosure, the following explanations of specific terms are provided:

Aptamer: The term aptamer, as used herein, refers to a non-naturallyoccurring nucleic acid that has a desirable action on a target molecule.A desirable action includes, but is not limited to, binding of thetarget, catalytically changing the target, reacting with the target in away that modifies or alters the target or the functional activity of thetarget, covalently attaching to the target (as in a suicide inhibitor),and facilitating the reaction between the target and another molecule.

Aptamer-Affinity Complex: As used herein, the terms “aptamer-targetaffinity complex”, “aptamer affinity complex” or “aptamer complex” or“complex” refer to a non-covalent complex that is formed by theinteraction of an aptamer with its target molecule. “Aptamer-targetaffinity complexes”, “aptamer affinity complexes” or “aptamer complexes”or “complexes” refer to more than one such set of complexes. Anaptamer-target affinity complex, aptamer affinity complex or aptamercomplex or complex can generally be reversed or dissociated by a changein an environmental condition, e.g., an increase in temperature, anincrease in salt concentration, or an addition of a denaturant. Ifdesired; however, such complexes may be a covalent interaction.

Amplifiable: The term amplifiable, as used herein, refers to a molecule(e.g., nucleic acid molecule or aptamer) that is capable of beingduplicated or copied to make more copies of the molecule.

Analog: The term analog, as used herein, refers to a structural chemicalanalog as well as a functional chemical analog. A structural chemicalanalog is a compound having a similar structure to another chemicalcompound but differing by one or more atoms or functional groups. Thisdifference may be a result of the addition of atoms or functionalgroups, absence of atoms or functional groups, the replacement of atomsor functional groups or a combination thereof. A functional chemicalanalog is a compound that has similar chemical, biochemical and/orpharmacological properties. The term analog may also encompass S and Rstereoisomers of a compound.

Bioactivity: The term bioactivity, as used herein, refers to one or moreintercellular, intracellular or extracellular process (e.g., cell-cellbinding, ligand-receptor binding, cell signaling, etc.) which can impactphysiological or pathophysiological processes.

Biological Sample: A biological sample, as used herein, refers to“sample”, and “test sample” are used interchangeably herein to refer toany material, biological fluid, tissue, or cell obtained or otherwisederived from an individual. This includes blood (including whole blood,leukocytes, peripheral blood mononuclear cells, buffy coat, plasma, andserum), sputum, tears, mucus, nasal washes, nasal aspirate, breath,urine, semen, saliva, meningeal fluid, amniotic fluid, glandular fluid,lymph fluid, nipple aspirate, bronchial aspirate, synovial fluid, jointaspirate, cells, a cellular extract, and cerebrospinal fluid. This alsoincludes experimentally separated fractions of all of the preceding. Forexample, a blood sample can be fractionated into serum or into fractionscontaining particular types of blood cells, such as red blood cells orwhite blood cells (leukocytes). If desired, a sample can be acombination of samples from an individual, such as a combination of atissue and fluid sample. The term “biological sample” also includesmaterials containing homogenized solid material, such as from a stoolsample, a tissue sample, or a tissue biopsy, for example. The term“biological sample” also includes materials derived from a tissueculture or a cell culture. Any suitable methods for obtaining abiological sample can be employed; exemplary methods include, e.g.,phlebotomy, swab (e.g., buccal swab), and a fine needle aspirate biopsyprocedure. Exemplary tissues susceptible to fine needle aspirationinclude lymph node, lung, lung washes, BAL (bronchoalveolar lavage),thyroid, breast, and liver. Samples can also be collected, e.g., bymicro dissection (e.g., laser capture micro dissection (LCM) or lasermicro dissection (LMD)), bladder wash, smear (e.g., a PAP smear), orductal lavage. A “biological sample” obtained or derived from anindividual includes any such sample that has been processed in anysuitable manner after being obtained from the individual.

C-5 Modified Pyrimidine: C-5 modified pyrimidine (or C-5 modifiednucleotide), as used herein, refers to a pyrimidine with a modificationat the C-5 position. Examples of a C-5 modified pyrimidine include thosedescribed in U.S. Pat. Nos. 5,719,273 and 5,945,527, as well as, U.S.Provisional Application Ser. No. 61/422,957, filed Dec. 14, 2010,entitled “Nuclease Resistant Oligonucleotides.” Additional examples areprovided herein.

Cell-Surface Protein: Cell-surface protein, as used herein, refers to aprotein that is expressed on the surface of a cell, cell membrane, cellwall envelope, or has a domain that is exposed on the outside surface ofthe cell, on the outside cell membrane or cell wall envelope withanother part or domain of the protein expressed within the cell membraneor cell wall envelope and/or in the intracellular space of a cell.

Consensus Sequence: Consensus sequence, as used herein, refers to anucleotide sequence that represents the most frequently observednucleotide found at each position of a series of nucleic acid sequencessubject to a sequence alignment.

Covalent Bond: Covalent bond or interaction refers to a chemical bondthat involves the sharing of at least a pair of electrons between atoms.

Enrich: The term enrich (or enrichment), as used herein, means tosubject a sample to a process such that the proportional representationof at least one component (e.g., the complex or aptamer-targetcomplexes) or group of components is resultantly enhanced compared toanother component or group of components. Enrich may mean to enrich onecomponent by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%compared to another component.

Inhibit: The term inhibit, as used herein, means to prevent or reducethe expression of a peptide or a polypeptide to an extent that thepeptide or polypeptide no longer has measurable activity or bioactivity;or to reduce the stability and/or reduce or prevent the activity of apeptide or a polypeptide to an extent that the peptide or polypeptide nolonger has measurable activity or bioactivity.

Microorganism: The term microorganism, as used herein, refers to asingle cell or multicellular organism and may include bacteria, archaea,protozoa, fungi, algae, microscopic plants, rotifers, planariansviruses.

Modified: The term modified (or modify or modification) and anyvariations thereof, when used in reference to an oligonucleotide, meansthat at least one of the four constituent nucleotide bases (i.e., A, G,T/U, and C) of the oligonucleotide is an analog or ester of a naturallyoccurring nucleotide.

Modulate: The term modulate, as used herein, means to alter theexpression level of a peptide, protein or polypeptide by increasing ordecreasing its expression level relative to a reference expressionlevel, and/or alter the stability and/or activity of a peptide, proteinor polypeptide by increasing or decreasing its stability and/or activitylevel relative to a reference stability and/or activity level.

Non-covalent Bond: Non-covalent bond or non-covalent interaction refersto a chemical bond or interaction that does not involve the sharing ofpairs of electrons between atoms. Examples of non-covalent bonds orinteractions include hydrogen bonds, ionic bonds (electrostatic bonds),van der Waals forces and hydrophobic interactions.

Nucleic Acid: Nucleic acid, as used herein, refers to any nucleic acidsequence containing DNA, RNA and/or analogs thereof and may includesingle, double and multi-stranded forms. The terms “nucleic acid”,“oligo”, “oligonucleotide” and “polynucleotide” may be usedinterchangeably.

Pharmaceutically Acceptable: Pharmaceutically acceptable, as usedherein, means approved by a regulatory agency of a federal or a stategovernment or listed in the U.S. Pharmacopoeia or other generallyrecognized pharmacopoeia for use in animals and, more particularly, inhumans.

Pharmaceutically Acceptable Salt: Pharmaceutically acceptable salt orsalt of a compound (e.g., aptamer), as used herein, refers to a productthat contains an ionic bond and is typically produced by reacting thecompound with either an acid or a base, suitable for administering to anindividual. A pharmaceutically acceptable salt can include, but is notlimited to, acid addition salts including hydrochlorides, hydrobromides,phosphates, sulfates, hydrogen sulfates, alkylsulfonates,arylsulfonates, arylalkylsulfonates, acetates, benzoates, citrates,maleates, fumarates, succinates, lactates, and tartrates; alkali metalcations such as Li, Na, K, alkali earth metal salts such as Mg or Ca, ororganic amine salts.

Pharmaceutical Composition: Pharmaceutical composition, as used herein,refers to formulation comprising an aptamer in a form suitable foradministration to an individual. A pharmaceutical composition istypically formulated to be compatible with its intended route ofadministration. Examples of routes of administration include, but arenot limited to, oral and parenteral, e.g., intravenous, intradermal,subcutaneous, inhalation, topical, transdermal, transmucosal, and rectaladministration.

SELEX: The term SELEX, as used herein, refers to generally to theselection for nucleic acids that interact with a target molecule in adesirable manner, for example binding with high affinity to a protein;and the amplification of those selected nucleic acids. SELEX may be usedto identify aptamers with high affinity to a specific target molecule.The term SELEX and “SELEX process” may be used interchangeably.

Sequence Identity: Sequence identity, as used herein, in the context oftwo or more nucleic acid sequences is a function of the number ofidentical nucleotide positions shared by the sequences (i.e., %identity=number of identical positions/total number of positions×100),taking into account the number of gaps, and the length of each gap thatneeds to be introduced to optimize alignment of two or more sequences.The comparison of sequences and determination of percent identitybetween two or more sequences can be accomplished using a mathematicalalgorithm, such as BLAST and Gapped BLAST programs at their defaultparameters (e.g., Altschul et al., J. Mol. Biol. 215:403, 1990; see alsoBLASTN at www.ncbi.nlm.nih.gov/BLAST). For sequence comparisons,typically one sequence acts as a reference sequence to which testsequences are compared. When using a sequence comparison algorithm, testand reference sequences are input into a computer, subsequencecoordinates are designated if necessary, and sequence algorithm programparameters are designated. The sequence comparison algorithm thencalculates the percent sequence identity for the test sequence(s)relative to the reference sequence, based on the designated programparameters. Optimal alignment of sequences for comparison can beconducted, e.g., by the local homology algorithm of Smith and Waterman,Adv. Appl. Math., 2:482, 1981, by the homology alignment algorithm ofNeedleman and Wunsch, J. Mol. Biol., 48:443, 1970, by the search forsimilarity method of Pearson and Lipman, Proc. Nat'l. Acad. Sci. USA85:2444, 1988, by computerized implementations of these algorithms (GAP,BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package,Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visualinspection (see generally, Ausubel, F. M. et al., Current Protocols inMolecular Biology, pub. by Greene Publishing Assoc. andWiley-Interscience (1987)). As used herein, when describing the percentidentity of a nucleic acid, such as a Spa (or SPA) aptamer, the sequenceof which is at least, for example, about 95% identical to a referencenucleotide sequence, it is intended that the nucleic acid sequence isidentical to the reference sequence except that the nucleic acidsequence may include up to five point mutations per each 100 nucleotidesof the reference nucleic acid sequence. In other words, to obtain adesired nucleic acid sequence, the sequence of which is at least about95% identical to a reference nucleic acid sequence, up to 5% of thenucleotides in the reference sequence may be deleted or substituted withanother nucleotide, or some number of nucleotides up to 5% of the totalnumber of nucleotides in the reference sequence may be inserted into thereference sequence (referred to herein as an insertion). These mutationsof the reference sequence to generate the desired sequence may occur atthe 5′ or 3′ terminal positions of the reference nucleotide sequence oranywhere between those terminal positions, interspersed eitherindividually among nucleotides in the reference sequence or in one ormore contiguous groups within the reference sequence.

Solid Support: Solid support refers to any substrate having a surface towhich molecules may be attached, directly or indirectly, through eithercovalent or non-covalent bonds. The solid support may include anysubstrate material that is capable of providing physical support for thecapture elements or probes that are attached to the surface. Thematerial is generally capable of enduring conditions related to theattachment of the capture elements or probes to the surface and anysubsequent treatment, handling, or processing encountered during theperformance of an assay. The materials may be naturally occurring,synthetic, or a modification of a naturally occurring material. Suitablesolid support materials may include silicon, a silicon wafer chip,graphite, mirrored surfaces, laminates, membranes, ceramics, plastics(including polymers such as, e.g., poly(vinyl chloride), cyclo-olefincopolymers, agarose gels or beads, polyacrylamide, polyacrylate,polyethylene, polypropylene, poly(4-methylbutene), polystyrene,polymethacrylate, poly(ethylene terephthalate), polytetrafluoroethylene(PTFE or Teflon®), nylon, poly(vinyl butyrate)), germanium, galliumarsenide, gold, silver, Langmuir Blodgett films, a flow through chip,etc., either used by themselves or in conjunction with other materials.Additional rigid materials may be considered, such as glass, whichincludes silica and further includes, for example, glass that isavailable as Bioglass. Other materials that may be employed includeporous materials, such as, for example, controlled pore glass beads,crosslinked beaded Sepharose® or agarose resins, or copolymers ofcrosslinked bis-acrylamide and azalactone. Other beads includenanoparticles, polymer beads, solid core beads, paramagnetic beads, ormicrobeads. Any other materials known in the art that are capable ofhaving one or more functional groups, such as any of an amino, carboxyl,thiol, or hydroxyl functional group, for example, incorporated on itssurface, are also contemplated.

The material used for a solid support may take any of a variety ofconfigurations ranging from simple to complex. The solid support canhave any one of a number of shapes, including a strip, plate, disk, rod,particle, bead, tube, well (microtiter), and the like. The solid supportmay be porous or non-porous, magnetic, paramagnetic, or non-magnetic,polydisperse or monodisperse, hydrophilic or hydrophobic. The solidsupport may also be in the form of a gel or slurry of closely-packed (asin a column matrix) or loosely-packed particles.

In one embodiment, the solid support with attached capture element isused to capture tagged aptamer-target affinity complexes oraptamer-target covalent complexes from a test mixture. In one particularexample, when the tag is a biotin moiety, the solid support could be astreptavidin-coated bead or resin such as Dynabeads M-280 Streptavidin,Dynabeads MyOne Streptavidin, Dynabeads M-270 Streptavidin (Invitrogen),Streptavidin Agarose Resin (Pierce), Streptavidin Ultralink Resin,MagnaBind Streptavidin Beads (ThermoFisher Scientific), BioMagStreptavidin, ProMag Streptavidin, Silica Streptavidin (BangsLaboratories), Streptavidin Sepharose High Performance (GE Healthcare),Streptavidin Polystyrene Microspheres (Microspheres-Nanospheres),Streptavidin Coated Polystyrene Particles (Spherotech), or any otherstreptavidin coated bead or resin commonly used by one skilled in theart to capture biotin-tagged molecules.

One object of the instant invention is to convert a protein signal intoan aptamer signal. As a result the quantity of aptamerscollected/detected is indicative of, and may be directly proportionalto, the quantity of target molecules bound and to the quantity of targetmolecules in the sample. A number of detection schemes can be employedwithout eluting the aptamer-target affinity or aptamer-target covalentcomplex from the second solid support after the second partitioning orcatch. In addition to the following embodiments of detection methods,other detection methods will be known to one skilled in the art.

Many detection methods require an explicit label to be incorporated intothe aptamer prior to detection. In these embodiments, labels, such as,for example, fluorescent or chemiluminescent dyes can be incorporatedinto aptamers either during or post synthesis using standard techniquesfor nucleic acid synthesis. Radioactive labels can be incorporatedeither during synthesis or post synthesis using standard enzymereactions with the appropriate reagents. Labeling can also occur afterthe second partitioning and elution by using suitable enzymatictechniques. For example, using a primer with the above mentioned labels,PCR will incorporate labels into the amplification product of the elutedaptamers. When using a gel technique for quantification, different sizemass labels can be incorporated using PCR as well. These mass labels canalso incorporate different fluorescent or chemiluminescent dyes foradditional multiplexing capacity. Labels may be added indirectly toaptamers by using a specific tag incorporated into the aptamer, eitherduring synthesis or post synthetically, and then adding a probe thatassociates with the tag and carries the label. The labels include thosedescribed above as well as enzymes used in standard assays forcolorimetric readouts, for example. These enzymes work in combinationwith enzyme substrates and include enzymes such as, for example,horseradish peroxidase (HRP) and alkaline phosphatase (AP). Labels mayalso include materials or compounds that are electrochemical functionalgroups for electrochemical detection.

For example, the aptamer may be labeled, as described above, with aradioactive isotope such as ³²P prior to contacting the test sample.Employing any one of the four basic assays, and variations thereof asdiscussed above, aptamer detection may be simply accomplished byquantifying the radioactivity on the second solid support at the end ofthe assay. The counts of radioactivity will be directly proportional tothe amount of target in the original test sample. Similarly, labeling anaptamer with a fluorescent dye, as described above, before contactingthe test sample allows for a simple fluorescent readout directly on thesecond solid support. A chemiluminescent label or a quantum dot can besimilarly employed for direct readout from the second solid support,requiring no aptamer elution.

In another embodiment, the amount or concentration of the aptamer-targetaffinity complex (or aptamer-target covalent complex) is determinedusing a “molecular beacon” during a replicative process (see, e.g.,Tyagi et al., Nat. Biotech. J. 6:49 53, 1998; U.S. Pat. No. 5,925,517).A molecular beacon is a specific nucleic acid probe that folds into ahairpin loop and contains a fluorophore on one end and a quencher on theother end of the hairpin structure such that little or no signal isgenerated by the fluorophore when the hairpin is formed. The loopsequence is specific for a target polynucleotide sequence and, uponhybridizing to the aptamer sequence the hairpin unfolds and therebygenerates a fluorescent signal.

For multiplexed detection of a small number of aptamers still bound tothe second solid support, fluorescent dyes with differentexcitation/emission spectra can be employed to detect and quantify two,or three, or five, or up to ten individual aptamers. Similarly differentsized quantum dots can be employed for multiplexed readouts. The quantumdots can be introduced after partitioning free aptamer from the secondsolid support. By using aptamer specific hybridization sequencesattached to unique quantum dots multiplexed readings for 2, 3, 4, 5, andup to 10 aptamers can be performed. Labeling different aptamers withdifferent radioactive isotopes that can be individually detected, suchas ³²P, ³H, ¹³C, and ³⁵S, can also be used for limited multiplexreadouts.

In one embodiment, a standard DNA hybridization array, or chip, is usedto hybridize each aptamer or photoaptamer to a unique or series ofunique probes immobilized on a slide or chip such as Agilent arrays,Illumina BeadChip Arrays, NimbleGen arrays or custom printed arrays.Each unique probe is complementary to a sequence on the aptamer. Thecomplementary sequence may be a unique hybridization tag incorporated inthe aptamer, or a portion of the aptamer sequence, or the entire aptamersequence. The aptamers released from the solid support after the secondpartitioning or catch are added to an appropriate hybridization bufferand processed using standard hybridization methods. For example, theaptamer solution is incubated for 12 hours with a DNA hybridizationarray at about 60° C. to ensure stringency of hybridization. The arraysare washed and then scanned in a fluorescent slide scanner, producing animage of the aptamer hybridization intensity on each feature of thearray. Image segmentation and quantification is accomplished using imageprocessing software, such as ArrayVision. In one embodiment, multiplexedaptamer assays can be detected using up to 25 aptamers, up to 50aptamers, up to 100 aptamers, up to 200 aptamers, up to 500 aptamers, upto 1000 aptamers, and up to 10,000 aptamers.

In one embodiment, addressable micro-beads having unique DNA probescomplementary to the aptamers as described above are used forhybridization. The micro-beads may be addressable with uniquefluorescent dyes, such as Luminex beads technology, or use bar codelabels as in the Illumina VeraCode technology, or laser poweredtransponders. In one embodiment, the aptamers released from the secondsolid support are added to an appropriate hybridization buffer andprocessed using standard micro-bead hybridization methods. For example,the aptamer solution is incubated for two hours with a set ofmicro-beads at about 60° C. to ensure stringency of hybridization. Thesolutions are then processed on a Luminex instrument which counts theindividual bead types and quantifies the aptamer fluorescent signal. Inanother embodiment, the VeraCode beads are contacted with the aptamersolution and hybridized for two hours at about 60° C. and then depositedon a gridded surface and scanned using a slide scanner foridentification and fluorescence quantification. In another embodiment,the transponder micro-beads are incubated with the aptamer sample atabout 60° C. and then quantified using an appropriate device for thetransponder micro-beads. In one embodiment, multiplex aptamer assays canbe detected by hybridization to micro-beads using up to 25 aptamers, upto 50 aptamers, up to 100 aptamers, up to 200 aptamers, and up to 500aptamers.

The sample containing the eluted aptamers can be processed toincorporate unique mass tags along with fluorescent labels as describedabove. The mass labeled aptamers are then injected into a CGEinstrument, essentially a DNA sequencer, and the aptamers are identifiedby their unique masses and quantified using fluorescence from the dyeincorporated during the labeling reaction. One exemplary example of thistechnique has been developed by Althea Technologies.

In many of the methods described above, the solution of aptamers can beamplified and optionally tagged before quantification. Standard PCRamplification can be used with the solution of aptamers eluted from thesecond solid support. Such amplification can be used prior to DNA arrayhybridization, micro-bead hybridization, and CGE readout.

In another embodiment, the aptamer-target affinity complex (oraptamer-target covalent complex) is detected and/or quantified usingQ-PCR. As used herein, “Q-PCR” refers to a PCR reaction performed insuch a way and under such controlled conditions that the results of theassay are quantitative, that is, the assay is capable of quantifying theamount or concentration of aptamer present in the test sample.

In one embodiment, the amount or concentration of the aptamer-targetaffinity complex (or aptamer-target covalent complex) in the test sampleis determined using TagMan® PCR. This technique generally relies on the5′-3′ exonuclease activity of the oligonucleotide replicating enzyme togenerate a signal from a targeted sequence. A TaqMan probe is selectedbased upon the sequence of the aptamer to be quantified and generallyincludes a 5′-end fluorophore, such as 6-carboxyfluorescein, forexample, and a 3′-end quencher, such as, for example, a6-carboxytetramethylfluorescein, to generate signal as the aptamersequence is amplified using polymerase chain reaction (PCR). As thepolymerase copies the aptamer sequence, the exonuclease activity freesthe fluorophore from the probe, which is annealed downstream from thePCR primers, thereby generating signal. The signal increases asreplicative product is produced. The amount of PCR product depends uponboth the number of replicative cycles performed as well as the startingconcentration of the aptamer.

In another embodiment, the amount or concentration of an aptamer-targetaffinity complex (or aptamer-target covalent complex) is determinedusing an intercalating fluorescent dye during the replicative process.The intercalating dye, such as, for example, SYBR® green, generates alarge fluorescent signal in the presence of double-stranded DNA ascompared to the fluorescent signal generated in the presence ofsingle-stranded DNA. As the double-stranded DNA product is formed duringPCR, the signal produced by the dye increases. The magnitude of thesignal produced is dependent upon both the number of PCR cycles and thestarting concentration of the aptamer.

In another embodiment, the aptamer-target affinity complex (oraptamer-target covalent complex) is detected and/or quantified usingmass spectrometry. Unique mass tags can be introduced using enzymatictechniques described above. For mass spectroscopy readout, no detectionlabel is required, rather the mass itself is used to both identify and,using techniques commonly used by those skilled in the art, quantifiedbased on the location and area under the mass peaks generated during themass spectroscopy analysis. An example using mass spectroscopy is theMassARRAY® system developed by Sequenom.

SOMAmer: The term SOMAmer (or SOMAmer reagent), as used herein, refersto an aptamer having improved off-rate characteristics. SOMAmer reagentsare alternatively referred to as Slow Off-Rate Modified Aptamers, andmay be selected via the improved SELEX methods described in U.S.Publication No. 20090004667, entitled “Method for Generating Aptamerswith Improved Off-Rates”, which is incorporated by reference in itsentirety. Slow Off-Rate Modified Aptamer refers to an aptamer (includingan aptamers comprising at least one nucleotide with a hydrophobicmodification) with an off-rate (t_(1/2)) of ≧30 minutes, ≧60 minutes,≧90 minutes, ≧120 minutes, ≧150 minutes, ≧180 minutes, ≧210 minutes, or≧240 minutes.

Spacer Sequence: Spacer sequence, as used herein, refers to any sequencecomprised of small molecule(s) covalently bound to the 5′-end, 3′-end orboth 5′ and 3′ ends of the nucleic acid sequence of an aptamer.Exemplary spacer sequences include, but are not limited to, polyethyleneglycols, hydrocarbon chains, and other polymers or copolymers thatprovide a molecular covalent scaffold connecting the consensus regionswhile preserving target-aptamer binding activity. In certain aspects,the spacer sequence may be covalently attached to the aptamer throughstandard linkages such as the terminal 3′ or 5′ hydroxyl, 2′ carbon, orbase modification such as the C5-position of pyrimidines, or C8 positionof purines.

Substantially Remove: Substantially remove, as used herein, means toremove at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80% or more of a component or components (e.g.,material not bound to the solid support) compared to another component(e.g., material bound to the solid support or aptamer-target complex).

Tag: As disclosed herein, an aptamer can further comprise a “tag,” whichrefers to a component that provides a means for attaching orimmobilizing an aptamer (and any target molecule that is bound to it) toa solid support and/or a means for detecting the aptamer or the complex(aptamer-target complex). A “tag” is a moiety that is capable ofassociating with a “capture element”. “Tags” or “capture elements”refers to more than one such set of components. The tag can be attachedto or included in the aptamer by any suitable method. Generally, the tagallows the aptamer to associate, either directly or indirectly, with acapture element or receptor that is attached to the solid support. Thecapture element is typically chosen (or designed) to be highly specificin its interaction with the tag and to retain that association duringsubsequent processing steps or procedures. A tag can enable thelocalization of an aptamer-target affinity complex (or covalentaptamer-target affinity complex) to a spatially defined address on asolid support. Different tags, therefore, can enable the localization ofdifferent aptamer-target covalent complexes to different spatiallydefined addresses on a solid support. A tag can be a polynucleotide, apolypeptide, a peptide nucleic acid, a locked nucleic acid, anoligosaccharide, a polysaccharide, an antibody, an affibody, an antibodymimic, a cell receptor, a ligand, a lipid, biotin, polyhistidine, or anyfragment or derivative of these structures, any combination of theforegoing, or any other structure with which a capture element (orlinker molecule, as described below) can be designed or configured tobind or otherwise associate with specificity. In the context of a tagfor detection purposes, the tag may be a dye, a quantum dot, aradiolabel, PCR primer sites, an electrochemical functional group, andan enzyme plus a detectable enzyme substrate. A tag may comprise twodistinct domains or regions that attach to the aptamer to allow theaptamer to be detected (e.g. PCR primer sites would include two distinctnucleic acid sequence that may attach to the 5′ or 3′ end of the aptameror in some cases where the one PCR primer site attaches to the 5′ end ofthe aptamer and the a second PCR primer set (of the pair) attach to the3′ end of the aptamer.

Generally, the tag may be added to the aptamer either pre- orpost-SELEX. In one embodiment, the tag is included on the 5′-end of theaptamer. In another embodiment, the tag is included on the 3′-end of theaptamer. In yet another embodiment, tags may be included on both the 3′and 5′ ends of the aptamers. In another embodiment, the tag may be aninternal segment of the aptamer.

Target Molecule: Target molecule (or target), as used herein, refers toany compound or molecule upon which a nucleic acid can act in adesirable manner (e.g., binding of the target, catalytically changingthe target, reacting with the target in a way that modifies or altersthe target or the functional activity of the target, covalentlyattaching to the target (as in a suicide inhibitor), and facilitatingthe reaction between the target and another molecule). Non-limitingexamples of a target molecule include a protein, peptide, nucleic acid,carbohydrate, lipid, polysaccharide, glycoprotein, hormone, receptor,antigen, antibody, virus, pathogen, toxic substance, substrate,metabolite, transition state analog, cofactor, inhibitor, drug, dye,nutrient, growth factor, cell, tissue, any portion or fragment of any ofthe foregoing, etc. Virtually any chemical or biological effector may bea suitable target. Molecules of any size can serve as targets. A targetcan also be modified in certain ways to enhance the likelihood orstrength of an interaction between the target and the nucleic acid. Atarget may also include any minor variation of a particular compound ormolecule, such as, in the case of a protein, for example, variations inits amino acid sequence, disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation ormodification, such as conjugation with a labeling component, which doesnot substantially alter the identity of the molecule. A “targetmolecule” or “target” is a set of copies of one type or species ofmolecule or multimolecular structure that is capable of binding to anaptamer. “Target molecules” or “targets” refer to more than one such setof molecules.

As used herein, the term “nucleotide” refers to a ribonucleotide or adeoxyribonucleotide, or a modified form thereof, as well as an analogthereof. Nucleotides include species that include purines (e.g.,adenine, hypoxanthine, guanine, and their derivatives and analogs) aswell as pyrimidines (e.g., cytosine, uracil, thymine, and theirderivatives and analogs).

As used herein, “nucleic acid,” “oligonucleotide,” and “polynucleotide”are used interchangeably to refer to a polymer of nucleotides andinclude DNA, RNA, DNA/RNA hybrids and modifications of these kinds ofnucleic acids, oligonucleotides and polynucleotides, wherein theattachment of various entities or moieties to the nucleotide units atany position are included. The terms “polynucleotide,”“oligonucleotide,” and “nucleic acid” include double- or single-strandedmolecules as well as triple-helical molecules. Nucleic acid,oligonucleotide, and polynucleotide are broader terms than the termaptamer and, thus, the terms nucleic acid, oligonucleotide, andpolynucleotide include polymers of nucleotides that are aptamers but theterms nucleic acid, oligonucleotide, and polynucleotide are not limitedto aptamers.

As used herein, the terms “modify”, “modified”, “modification”, and anyvariations thereof, when used in reference to an oligonucleotide, meansthat at least one of the four constituent nucleotide bases (i.e., A, G,T/U, and C) of the oligonucleotide is an analog or ester of a naturallyoccurring nucleotide. In some embodiments, the modified nucleotideconfers nuclease resistance to the oligonucleotide. In some embodiments,the modified nucleotides lead to predominantly hydrophobic interactionsof aptamers with protein targets resulting in high binding efficiencyand stable co-crystal complexes. A pyrimidine with a substitution at theC-5 position is an example of a modified nucleotide. Modifications caninclude backbone modifications, methylations, unusual base-pairingcombinations such as the isobases isocytidine and isoguanidine, and thelike. Modifications can also include 3′ and 5′ modifications, such ascapping. Other modifications can include substitution of one or more ofthe naturally occurring nucleotides with an analog, internucleotidemodifications such as, for example, those with uncharged linkages (e.g.,methyl phosphonates, phosphotriesters, phosphoamidates, carbamates,etc.) and those with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those with intercalators (e.g., acridine,psoralen, etc.), those containing chelators (e.g., metals, radioactivemetals, boron, oxidative metals, etc.), those containing alkylators, andthose with modified linkages (e.g., alpha anomeric nucleic acids, etc.).Further, any of the hydroxyl groups ordinarily present on the sugar of anucleotide may be replaced by a phosphonate group or a phosphate group;protected by standard protecting groups; or activated to prepareadditional linkages to additional nucleotides or to a solid support. The5′ and 3′ terminal OH groups can be phosphorylated or substituted withamines, organic capping group moieties of from about 1 to about 20carbon atoms, polyethylene glycol (PEG) polymers in some embodimentsranging from about 10 to about 80 kDa, PEG polymers in some embodimentsranging from about 20 to about 60 kDa, or other hydrophilic orhydrophobic biological or synthetic polymers. In some embodiments,modifications are of the C-5 position of pyrimidines. Thesemodifications can be produced through an amide linkage directly at theC-5 position or by other types of linkages.

Polynucleotides can also contain analogous forms of ribose ordeoxyribose sugars that are generally known in the art, including2′-O-methyl-, 2′-O-allyl, 2′-fluoro- or 2′-azido-ribose, carbocyclicsugar analogs, α-anomeric sugars, epimeric sugars such as arabinose,xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses,acyclic analogs and abasic nucleoside analogs such as methyl riboside.As noted above, one or more phosphodiester linkages may be replaced byalternative linking groups. These alternative linking groups includeembodiments wherein phosphate is replaced by P(O)S (“thioate”), P(S)S(“dithioate”), (O)NR₂ (“amidate”), P(O)R, P(O)OR′, CO or CH₂(“formacetal”), in which each R or R′ is independently H or substitutedor unsubstituted alkyl (1-20 C) optionally containing an ether (—O—)linkage, aryl, alkenyl, cycloalky, cycloalkenyl or araldyl. Not alllinkages in a polynucleotide need be identical. Substitution ofanalogous forms of sugars, purines, and pyrimidines can be advantageousin designing a final product, as can alternative backbone structureslike a polyamide backbone, for example.

As used herein, the term “nuclease” refers to an enzyme capable ofcleaving the phosphodiester bond between nucleotide subunits of anoligonucleotide. As used herein, the term “endonuclease” refers to anenzyme that cleaves phosphodiester bond(s) at a site internal to theoligonucleotide. As used herein, the term “exonuclease” refers to anenzyme which cleaves phosphodiester bond(s) linking the end nucleotidesof an oligonucleotide. Biological fluids typically contain a mixture ofboth endonucleases and exonucleases.

As used herein, the terms “nuclease resistant” and “nuclease resistance”refers to the reduced ability of an oligonucleotide to serve as asubstrate for an endo- or exonuclease, such that, when contacted withsuch an enzyme, the oligonucleotide is either not degraded or isdegraded more slowly than an oligonucleotide composed of unmodifiednucleotides.

As used herein, the term “at least one pyrimidine,” when referring tomodifications of a nucleic acid, refers to one, several, or allpyrimidines in the nucleic acid, indicating that any or all occurrencesof any or all of C, T, or U in a nucleic acid may be modified or not.

As used herein, A, C, G, U and T denote dA, dC, dG, dU and dTrespectively, unless otherwise specified.

As used herein, “nucleic acid ligand,” “aptamer,” and “clone” are usedinterchangeably to refer to a non-naturally occurring nucleic acid thathas a desirable action on a target molecule. A desirable actionincludes, but is not limited to, binding of the target, catalyticallychanging the target, reacting with the target in a way that modifies oralters the target or the functional activity of the target, covalentlyattaching to the target (as in a suicide inhibitor), and facilitatingthe reaction between the target and another molecule. In someembodiments, the action is specific binding affinity for a targetmolecule, such target molecule being a three dimensional chemicalstructure other than a polynucleotide that binds to the nucleic acidligand through a mechanism which is independent of Watson/Crick basepairing or triple helix formation, wherein the aptamer is not a nucleicacid having the known physiological function of being bound by thetarget molecule. Aptamers to a given target include nucleic acids thatare identified from a candidate mixture of nucleic acids, where theaptamer is a ligand of the target, by a method comprising: (a)contacting the candidate mixture with the target, wherein nucleic acidshaving an increased affinity to the target relative to other nucleicacids in the candidate mixture can be partitioned from the remainder ofthe candidate mixture; (b) partitioning the increased affinity nucleicacids from the remainder of the candidate mixture; and (c) amplifyingthe increased affinity nucleic acids to yield a ligand-enriched mixtureof nucleic acids, whereby aptamers of the target molecule areidentified. It is recognized that affinity interactions are a matter ofdegree; however, in this context, the “specific binding affinity” of anaptamer for its target means that the aptamer binds to its targetgenerally with a much higher degree of affinity than it binds to other,non-target, components in a mixture or sample. An “aptamer” or “nucleicacid ligand” is a set of copies of one type or species of nucleic acidmolecule that has a particular nucleotide sequence. An aptamer caninclude any suitable number of nucleotides. “Aptamers” refer to morethan one such set of molecules. Different aptamers can have either thesame or different numbers of nucleotides. Aptamers may be DNA or RNA andmay be single stranded, double stranded, or contain double stranded ortriple stranded regions.

As used herein, “protein” is used synonymously with “peptide,”“polypeptide,” or “peptide fragment.” A “purified” polypeptide, protein,peptide, or peptide fragment is substantially free of cellular materialor other contaminating proteins from the cell, tissue, or cell-freesource from which the amino acid sequence is obtained, or substantiallyfree from chemical precursors or other chemicals when chemicallysynthesized.

A “SPA aptamer” is an aptamer that is capable of binding to the SPAprotein. The term “SpA”, “SPA” or “Spa” may be used interchangeably torefer to the SPA protein or SPA aptamer.

A “ClfA aptamer” is an aptamer that is capable of binding to the ClfAprotein.

A “ClfB aptamer” is an aptamer that is capable of binding to the ClfBprotein.

A “FnbA aptamer” is an aptamer that is capable of binding to the FnbAprotein.

A “FnbB aptamer” is an aptamer that is capable of binding to the FnbBprotein.

A “IsdA aptamer” is an aptamer that is capable of binding to the IsdAprotein.

A “IsdB aptamer” is an aptamer that is capable of binding to the IsdBprotein.

A “IsdC aptamer” is an aptamer that is capable of binding to the IsdCprotein.

A “IsdH aptamer” is an aptamer that is capable of binding to the IsdHprotein.

A “SasD aptamer” is an aptamer that is capable of binding to the SasDprotein.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. “Comprising A or B” means including A or B, or Aand B. It is further to be understood that all base sizes or amino acidsizes, and all molecular weight or molecular mass values, given fornucleic acids or polypeptides are approximate, and are provided fordescription.

Further, ranges provided herein are understood to be shorthand for allof the values within the range. For example, a range of 1 to 50 isunderstood to include any number, combination of numbers, or sub-rangefrom the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or50 (as well as fractions thereof unless the context clearly dictatesotherwise). Any concentration range, percentage range, ratio range, orinteger range is to be understood to include the value of any integerwithin the recited range and, when appropriate, fractions thereof (suchas one tenth and one hundredth of an integer), unless otherwiseindicated. Also, any number range recited herein relating to anyphysical feature, such as polymer subunits, size or thickness, are to beunderstood to include any integer within the recited range, unlessotherwise indicated. As used herein, “about” or “consisting essentiallyof mean±20% of the indicated range, value, or structure, unlessotherwise indicated. As used herein, the terms “include” and “comprise”are open ended and are used synonymously. It should be understood thatthe terms “a” and “an” as used herein refer to “one or more” of theenumerated components. The use of the alternative (e.g., “or”) should beunderstood to mean either one, both, or any combination thereof of thealternatives

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present disclosure,suitable methods and materials are described below. All publications,patent applications, patents, and other references mentioned herein areincorporated by reference in their entirety. In case of conflict, thepresent specification, including explanations of terms, will control. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting.

Overview

Binding agents to specific components on the surface of microorganismscan be valuable diagnostic tools useful for different detectionplatforms.

SELEX

SELEX generally includes preparing a candidate mixture of nucleic acids,binding of the candidate mixture to the desired target molecule to forman affinity complex, separating the affinity complexes from the unboundcandidate nucleic acids, separating and isolating the nucleic acid fromthe affinity complex, purifying the nucleic acid, and identifying aspecific aptamer sequence. The process may include multiple rounds tofurther refine the affinity of the selected aptamer. The process caninclude amplification steps at one or more points in the process. See,e.g., U.S. Pat. No. 5,475,096, entitled “Nucleic Acid Ligands”. TheSELEX process can be used to generate an aptamer that covalently bindsits target as well as an aptamer that non-covalently binds its target.See, e.g., U.S. Pat. No. 5,705,337 entitled “Systematic Evolution ofNucleic Acid Ligands by Exponential Enrichment: Chemi-SELEX.”

The SELEX process can be used to identify high-affinity aptamerscontaining modified nucleotides that confer improved characteristics onthe aptamer, such as, for example, improved in vivo stability orimproved delivery characteristics. Examples of such modificationsinclude chemical substitutions at the ribose and/or phosphate and/orbase positions. SELEX process-identified aptamers containing modifiednucleotides are described in U.S. Pat. No. 5,660,985, entitled “HighAffinity Nucleic Acid Ligands Containing Modified Nucleotides,” whichdescribes oligonucleotides containing nucleotide derivatives chemicallymodified at the 5′- and 2′-positions of pyrimidines. U.S. Pat. No.5,580,737, see supra, describes highly specific aptamers containing oneor more nucleotides modified with 2′-amino (2′-NH₂), 2′-fluoro (2′-F),and/or 2′-O-methyl (2′-OMe). See also, U.S. Patent ApplicationPublication 20090098549, entitled “SELEX and PHOTOSELEX”, whichdescribes nucleic acid libraries having expanded physical and chemicalproperties and their use in SELEX and photoSELEX.

SELEX can also be used to identify aptamers that have desirable off-ratecharacteristics. See U.S. Patent Application Publication 20090004667,entitled “Method for Generating Aptamers with Improved Off-Rates,” whichdescribes improved SELEX methods for generating aptamers that can bindto target molecules. As mentioned above, these slow off-rate aptamersare known as “SOMAmers.” Methods for producing aptamers or SOMAmerreagents and photoaptamers or SOMAmer reagents having slower rates ofdissociation from their respective target molecules are described. Themethods involve contacting the candidate mixture with the targetmolecule, allowing the formation of nucleic acid-target complexes tooccur, and performing a slow off-rate enrichment process wherein nucleicacid-target complexes with fast dissociation rates will dissociate andnot reform, while complexes with slow dissociation rates will remainintact. Additionally, the methods include the use of modifiednucleotides in the production of candidate nucleic acid mixtures togenerate aptamers or SOMAmer reagents with improved off-rateperformance.

A variation of this assay employs aptamers that include photoreactivefunctional groups that enable the aptamers to covalently bind or“photocrosslink” their target molecules. See, e.g., U.S. Pat. No.6,544,776 entitled “Nucleic Acid Ligand Diagnostic Biochip.” Thesephotoreactive aptamers are also referred to as photoaptamers. See, e.g.,U.S. Pat. No. 5,763,177, U.S. Pat. No. 6,001,577, and U.S. Pat. No.6,291,184, each of which is entitled “Systematic Evolution of NucleicAcid Ligands by Exponential Enrichment: Photoselection of Nucleic AcidLigands and Solution SELEX”; see also, e.g., U.S. Pat. No. 6,458,539,entitled “Photoselection of Nucleic Acid Ligands.” After the microarrayis contacted with the sample and the photoaptamers have had anopportunity to bind to their target molecules, the photoaptamers arephotoactivated, and the solid support is washed to remove anynon-specifically bound molecules. Harsh wash conditions may be used,since target molecules that are bound to the photoaptamers are generallynot removed, due to the covalent bonds created by the photoactivatedfunctional group(s) on the photoaptamers.

In both of these assay formats, the aptamers or SOMAmer reagents areimmobilized on the solid support prior to being contacted with thesample. Under certain circumstances, however, immobilization of theaptamers or SOMAmer reagents prior to contact with the sample may notprovide an optimal assay. For example, pre-immobilization of theaptamers or SOMAmer reagents may result in inefficient mixing of theaptamers or SOMAmer reagents with the target molecules on the surface ofthe solid support, perhaps leading to lengthy reaction times and,therefore, extended incubation periods to permit efficient binding ofthe aptamers or SOMAmer reagents to their target molecules. Further,when photoaptamers or photoaptamers are employed in the assay anddepending upon the material utilized as a solid support, the solidsupport may tend to scatter or absorb the light used to effect theformation of covalent bonds between the photoaptamers or photoaptamersand their target molecules. Moreover, depending upon the methodemployed, detection of target molecules bound to their aptamers orphotoaptamers can be subject to imprecision, since the surface of thesolid support may also be exposed to and affected by any labeling agentsthat are used. Finally, immobilization of the aptamers or SOMAmerreagents on the solid support generally involves an aptamer or SOMAmerreagent-preparation step (i.e., the immobilization) prior to exposure ofthe aptamers or SOMAmer reagents to the sample, and this preparationstep may affect the activity or functionality of the aptamers or SOMAmerreagents.

Aptamer assays that permit an aptamer to capture its target in solutionand then employ separation steps that are designed to remove specificcomponents of the aptamer-target mixture prior to detection have alsobeen described (see U.S. Patent Application Publication 20090042206,entitled “Multiplexed Analyses of Test Samples”). The described aptamerassay methods enable the detection and quantification of a non-nucleicacid target (e.g., a protein target) in a test sample by detecting andquantifying a nucleic acid (i.e., a aptamer). The described methodscreate a nucleic acid surrogate (i.e., the aptamer) for detecting andquantifying a non-nucleic acid target, thus allowing the wide variety ofnucleic acid technologies, including amplification, to be applied to abroader range of desired targets, including protein targets.

Embodiments of the SELEX process in which the target is a peptide aredescribed in U.S. Pat. No. 6,376,190, entitled “Modified SELEX ProcessesWithout Purified Protein.”

Chemical Modifications to Aptamers

Aptamers may contain modified nucleotides that improve it properties andcharacteristics. Non-limiting examples of such improvements include, invivo stability, stability against degradation, binding affinity for itstarget, and/or improved delivery characteristics.

Examples of such modifications include chemical substitutions at theribose and/or phosphate and/or base positions of a nucleotide. SELEXprocess-identified aptamers containing modified nucleotides aredescribed in U.S. Pat. No. 5,660,985, entitled “High Affinity NucleicAcid Ligands Containing Modified Nucleotides,” which describesoligonucleotides containing nucleotide derivatives chemically modifiedat the 5′- and 2′-positions of pyrimidines. U.S. Pat. No. 5,580,737, seesupra, describes highly specific aptamers containing one or morenucleotides modified with 2′-amino (2′-NH₂), 2′-fluoro (2′-F), and/or2′-O-methyl (2′-OMe). See also, U.S. Patent Application Publication No.20090098549, entitled “SELEX and PHOTOSELEX,” which describes nucleicacid libraries having expanded physical and chemical properties andtheir use in SELEX and photoSELEX.

Specific examples of a C-5 modification include substitution ofdeoxyuridine at the C-5 position with a substituent independentlyselected from: benzylcarboxyamide (alternatively benzylaminocarbonyl)(Bn), naphthylmethylcarboxyamide (alternativelynaphthylmethylaminocarbonyl) (Nap), tryptaminocarboxyamide(alternatively tryptaminocarbonyl) (Trp), and isobutylcarboxyamide(alternatively isobutylaminocarbonyl) (iBu) as illustrated immediatelybelow.

Specific examples of a C-5 modification include substitution ofdeoxyuridine Chemical modifications of a C-5 modified pyrimidine canalso be combined with, singly or in any combination, 2′-position sugarmodifications, modifications at exocyclic amines, and substitution of4-thiouridine and the like.

Representative C-5 modified pyrimidines include:5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU),5-(N-benzylcarboxyamide)-2′-O-methyluridine,5-(N-benzylcarboxyamide)-2′-fluorouridine,5-(N-isobutylcarboxyamide)-2′-deoxyuridine (iBudU),5-(N-isobutylcarboxyamide)-2′-O-methyluridine,5-(N-isobutylcarboxyamide)-2′-fluorouridine,5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU),5-(N-tryptaminocarboxyamide)-2′-O-methyluridine,5-(N-tryptaminocarboxyamide)-2′-fluorouridine,5-(N-[1-(3-trimethylammonium) propyl] carboxyamide)-2′-deoxyuridinechloride, 5-(N-naphthylmethylcarboxyamide)-2′-deoxyuridine (NapdU),5-(N-naphthylmethylcarboxyamide)-2′-O-methyluridine,5-(N-naphthylmethylcarboxyamide)-2′-fluorouridine or5-(N-[1-(2,3-dihydroxypropyl)]carboxyamide)-2′-deoxyuridine).

If present, a modification to the nucleotide structure can be impartedbefore or after assembly of the polynucleotide. A sequence ofnucleotides can be interrupted by non-nucleotide components. Apolynucleotide can be further modified after polymerization, such as byconjugation with a labeling component.

Additional non-limiting examples of modified nucleotides (e.g., C-5modified pyrimidine) that may be incorporated into the nucleic acidsequences of the present disclosure include the following:

wherein R′ is defined as follows:

whereinR″″ is selected from the group consisting of a branched or linear loweralkyl (C1-C20); hydroxyl (OH), halogen (F, Cl, Br, I); nitrile (CN);boronic acid (BO₂H₂); carboxylic acid (COOH); carboxylic acid ester(COOR″); primary amide (CONH₂); secondary amide (CONHR″); tertiary amide(CONR″R′″); sulfonamide (SO₂NH₂); N-alkylsulfonamide (SONHR″);whereinR″, R′″ are independently selected from a group consisting of a branchedor linear lower alkyl (C1-C2)); phenyl (C6H5); an R″″ substituted phenylring (R″″C6H4); wherein R″″ is defined above; a carboxylic acid (COOH);a carboxylic acid ester (COOR); wherein R is a branched or linear loweralkyl (C1-C20); and cycloalkyl; wherein R″═R′″═(CH2)n;wherein n=2-10.

Further, C-5 modified pyrimidine nucleotides include the following:

In some embodiments, the modified nucleotide confers nuclease resistanceto the oligonucleotide. A pyrimidine with a substitution at the C-5position is an example of a modified nucleotide. Modifications caninclude backbone modifications, methylations, unusual base-pairingcombinations such as the isobases isocytidine and isoguanidine, and thelike. Modifications can also include 3′ and 5′ modifications, such ascapping. Other modifications can include substitution of one or more ofthe naturally occurring nucleotides with an analog, internucleotidemodifications such as, for example, those with uncharged linkages (e.g.,methyl phosphonates, phosphotriesters, phosphoamidates, carbamates,etc.) and those with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those with intercalators (e.g., acridine,psoralen, etc.), those containing chelators (e.g., metals, radioactivemetals, boron, oxidative metals, etc.), those containing alkylators, andthose with modified linkages (e.g., alpha anomeric nucleic acids, etc.).Further, any of the hydroxyl groups ordinarily present on the sugar of anucleotide may be replaced by a phosphonate group or a phosphate group;protected by standard protecting groups; or activated to prepareadditional linkages to additional nucleotides or to a solid support. The5′ and 3′ terminal OH groups can be phosphorylated or substituted withamines, organic capping group moieties of from about 1 to about 20carbon atoms, polyethylene glycol (PEG) polymers in one embodimentranging from about 10 to about 80 kDa, PEG polymers in anotherembodiment ranging from about 20 to about 60 kDa, or other hydrophilicor hydrophobic biological or synthetic polymers. In one embodiment,modifications are of the C-5 position of pyrimidines. Thesemodifications can be produced through an amide linkage directly at theC-5 position or by other types of linkages.

Polynucleotides can also contain analogous forms of ribose ordeoxyribose sugars that are generally known in the art, including2′-O-methyl-, 2′-O-allyl, 2′-fluoro- or 2′-azido-ribose, carbocyclicsugar analogs, a-anomeric sugars, epimeric sugars such as arabinose,xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses,acyclic analogs and abasic nucleoside analogs such as methyl riboside.As noted above, one or more phosphodiester linkages may be replaced byalternative linking groups. These alternative linking groups includeembodiments wherein phosphate is replaced by P(O)S (“thioate”), P(S)S(“dithioate”), (O)NR₂ (“amidate”), P(O)R, P(O)OR′, CO or CH₂(“formacetal”), in which each R or R′ is independently H or substitutedor unsubstituted alkyl (1-20 C) optionally containing an ether (—O—)linkage, aryl, alkenyl, cycloalky, cycloalkenyl or araldyl. Not alllinkages in a polynucleotide need be identical. Substitution ofanalogous forms of sugars, purines, and pyrimidines can be advantageousin designing a final product, as can alternative backbone structureslike a polyamide backbone, for example.

The following examples are provided to illustrate certain particularfeatures and/or embodiments. These examples should not be construed tolimit the disclosure to the particular features or embodimentsdescribed.

Staphylococcus aureus (Also Referred to Herein as S. aureus)

Staining of surface antigens for immunofluorescence microscopy has beendemonstrated using antibody-fluorophore conjugates to detect relativelylow numbers of S. aureus cells over time in in vivo infection models(Timofeyeva et al., 2014). Short peptides as specific ligands to the S.aureus cell surface have been identified by phage-display, and asynthetic consensus peptide (SA5-1) was able to detect approximately 100CFU ml⁻¹ in a spiked biological sample using fluorescent quantum dots(Rao et al., 2013).

Major groups are the MSCRAMMs (Microbial surface components recognizingadhesive matrix molecules), the SERAMs (secretable expanded repertoireadhesive molecules), as well as other extracellular toxins and immuneevasion factors (Gill et al., 2005; Speziale et al., 2009). It ispossible to use whole bacterial cells for SELEX (Cao et al., 2009), orsurface-associated proteins extracted from cells with LiCl, lysostaphin,or 2% SDS (Palma et al., 1998; Hussain et al., 2001; Roche et al.,2003), or released by trypsin-shaving (Ythier et al., 2012). However,the composition of the surface proteome in vitro varies betweendifferent strains and depends on media and growth phase. Furthermore,staphylococci other than S. aureus express closely related proteins,which may hamper the isolation of species-specific reagents withoutcareful counter-selection. Therefore, we chose to focus onwell-conserved S. aureus-specific cell surface proteins that are knownto be expressed in abundance and under most growth conditions, andproduced these SELEX targets in recombinant form.

Proteins that are exposed on the S. aureus cell surface can directlyinteract with extracellular molecules, including drugs and antibodies,and these adhesions or immune evasion proteins represent vaccinecandidate targets (Stranger-Jones et al., 2006; McCarthy and Lindsay,2010; Dreisbach et al., 2011). The S. aureus cell envelope, cellwall-associated proteins and mechanisms for protein attachment, arequite well understood (Dreisbach et al., 2011). Comparison of wholegenome sequences of 58 S. aureus strains, however, revealed variationsin proteins implicated in adhesion or immune response evasion, orproteins that were missing or truncated in certain strains (McCarthy andLindsay, 2010). Adhesins include a family of surface proteins covalentlyattached to the peptidoglycan via a conserved LPXTG motif (Schneewind etal., 1995). Proteomic and transcriptomic profiling of surface proteinshas been shown to correlate well with adherence-phenotypes in S. aureus(Roche et al., 2003; Ythier et al., 2012).

The ten surface-associated proteins for which we generated aptamersinclude SpA, ClfA, ClfB, FnbA, FnbB, SasD, IsdA, IsdB, IsdC, and IsdH.All of these proteins are attached to the cell-wall via sortase-mediatedcleavage between the threonine and the glycine of the LPXTG sortasemotif and become amide-linked to the pentaglycine cross-bridge ofpeptidoglycan (Marraffini et al., 2006). Since our goal was to obtainbinding agents to S. aureus cells, we produced recombinant proteins thatrepresent the surface-exposed domains but lack the signal sequences andthe repeat regions of the cell wall-embedded domain. S. aureus protein A(SpA) is present on the bacterial surface as well as secreted into theextracellular milieu. SpA is a potent immune evasion factor since itbinds the Fc region of antibodies and the Fab regions of the B-cellreceptor (IgM), thus blocking opsonophagocytosis and causing B-celldeath (Falugi et al., 2013; Kobayashi and Deleo, 2013). Since SpAwell-conserved in among S. aureus but is absent in non-pathogenicstaphylococci such as Staphylococcus epidermidis and Staphylococcushaemolyticus, this protein represents an attractive diagnostic target.Clinical isolates with truncated SpA variants have been described thatlack the XC region with the C-terminal sorting signal and are thus foundmainly extracellular (Sorum et al., 2013). ClfA and ClfB arestructurally related fibrinogen-binding proteins (McDevitt et al., 1997;Ni Eidhin et al., 1998). ClfB is one of the key factors responsible foradherence to desquamated epithelial cells of the anterior nares, and istypically produced during early exponential phase of growth (Ni Eidhinet al., 1998). FnbA and FnbB adhere to components of the extracellularmatrix, both fibronectin and elastin, and are important for colonizationof host tissues during infection (Roche et al., 2004). SasD is aputative adhesion protein with unknown physiological role (Roche et al.,2003; Ythier et al., 2012). Four of the proteins belong to theiron-responsive surface determinant (Isd) system that is induced in S.aureus under iron-limiting conditions and is important for capture ofheme from hemoglobin (IsdB, IsdH) and its transport (IsdA, IsdC) acrossthe cell wall (Mazmanian et al., 2003; Grigg et al., 2010).

As a proof-of-concept and to assess their efficiency, the aptamersgenerated against S. aureus cell surface-associated proteins were usedto capture and detect S. aureus using qPCR and also to directly detectthe cells by flow cytometry.

SOMAmer (slow off-rate modified aptamer) reagents are made fromsingle-stranded DNA (ssDNA) that contain pyrimidine residues modified attheir 5-prime position with mimics of amino acid side-chains and havequite long (>30 min) dissociation rates (Gold et al., 2010). Thesefeatures lead to better affinity and better kinetic properties ofaptamers compared to standard RNA or DNA aptamers. Virtually any proteincan be used for SELEX (systematic evolution of ligands by exponentialenrichment) to generate specific, high-affinity aptamers in multiplerounds of selection with kinetic challenge, partitioning, andamplification from a random library of modified ssDNA (Gold et al.,2010; Vaught et al., 2010). Advantages of aptamers over antibodiesinclude exceptional thermostability in solution, lower molecular weight,higher multiplexing capabilities, chemical stability to heat, drying,and solvents, reversible renaturation, ease of reagent manufacturing,consistent lot-to-lot performance and lower cost. Aptamers have beengenerated to >1000 human proteins and are the basis for the SOMAscan™proteomic platform developed by SomaLogic to measure these proteinssimultaneously and with high accuracy in a small (0.1 ml) blood sample.The application of this highly multiplexed assay has led to thediscovery of biomarkers in various areas of medicine (Gold et al.,2012). With respect to microbial proteins, we have previously reportedon the characterization of aptamers for Clostridium difficile toxins andshown the wide range of potential applications of these binding agents(Ochsner et al., 2013).

Slow off-rate modified aptamer (SOMAmer reagent) reagents were generatedto several Staphylococcus aureus cell surface-associated proteins viaSELEX with multiple modified DNA libraries using purified recombinant ornative proteins. High-affinity binding agents with sub-nanomolar K_(d)'swere obtained for staphylococcal protein A (SpA), clumping factors(ClfA, ClfB), fibronectin-binding proteins (FnbA, FnbB) andiron-regulated surface determinants (Isd). Further screening revealedseveral aptamers that specifically bound to S. aureus cells from allstrains that were tested, but not to other staphylococci or otherbacteria. SpA and ClfA aptamers proved useful for the selective captureand enrichment of S. aureus cells from low cell-density matrices, asshown by culture and PCR, leading to improved limits of detection andefficient removal of PCR inhibitors. Detection of S. aureus cells wasenhanced by several orders of magnitude when the bacterial cell surfacewas coated with aptamers followed by qPCR of the aptamers. Furthermore,fluorescence labeled SpA aptamers demonstrated their utility as directdetection agents in flow cytometry.

Kits Comprising Aptamer Compositions

The present disclosure provides kits comprising any of the aptamersdescribed herein. Such kits can comprise, for example, (1) at least oneaptamer that binds a target; and (2) at least one pharmaceuticallyacceptable carrier, such as a solvent or solution. Additional kitcomponents can optionally include, for example: (1) any of thepharmaceutically acceptable excipients identified herein, such asstabilizers, buffers, etc., (2) at least one container, vial or similarapparatus for holding and/or mixing the kit components; and (3) deliveryapparatus.

In another aspect this disclosure provides an aptamer sequence thatbinds the SPA protein represented by SEQ ID NO: 9. The nucleotidesequence may be further generalized to the following sequence:

(SEQ ID NO: 14) GGCWWCGGGWACCWAWWAWNGGWWWAGCC(N)_(n)GWCwherein “W” in the sequence represents a position that may be occupiedby a C-5 modified pyrimidine, and “N” represents a position that may beoccupied by any unmodified or modified nucleotide, and n is from 0 to 2(or 0, 1 or 2).

In another aspect, N is a C, T, G or A. In another aspect, N is a C, Tor A.

In another aspect, the nucleotide sequence may include up to about 100nucleotides, up to about 95 nucleotides, up to about 90 nucleotides, upto about 85 nucleotides, up to about 80 nucleotides, up to about 75nucleotides, up to about 70 nucleotides, up to about 65 nucleotides, upto about 60 nucleotides, up to about 55 nucleotides, up to about 50nucleotides, up to about 45 nucleotides, up to about 40 nucleotides.

In another aspect this disclosure, the aptamer may be at least about 95%identical, at least about 90% identical, at least about 85% identical,at least about 80% identical, or at least about 75% identical to any ofSEQ ID NO:14. In another embodiment, the aptamer includes a sequencefragments of SEQ ID NO:14.

In another aspect, the aptamer comprises from 1 to 50 (or 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49 or 50) C-5 modified pyrimidines. In anotheraspect, the aptamer comprises from 5 to 30 (or 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29or 30) C-5 modified pyrimidines. In another aspect, the aptamercomprises from 10 to 15 (or from 10, 11, 12, 13, 14 or 15) C-5 modifiedpyrimidines.

In another aspect, the aptamer comprises from about 1% to 100% (or 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99 or 100%) C-5 modified pyrimidines. In another aspect, theaptamer comprises from about 10% to about 50% (or 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50%)C-5 modified pyrimidines. In another aspect, the aptamer comprises, fromabout 20% to about 40% (or 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39 or 40%) C-5 modified pyrimidines. Inanother aspect, the aptamer comprises from about 25% to about 35% (or25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35%) C-5 modified pyrimidines.In another aspect, the aptamer comprises from about 27% to about 33% (or27, 28, 29, 30, 31, 32 or 33%) C-5 modified pyrimidines. In anotheraspect, the aptamer comprises from about 37% to about 43% (or 37, 38,39, 40, 41, 42, 43%) C-5 modified pyrimidines.

In another aspect, W may represent a C-5 modified uridine or cytidine.

In another aspect, W may represent a C-5 modified pyrimidine illustratedimmediately below:

wherein R′ is defined as follows:

whereinR″″ is selected from the group consisting of a branched or linear loweralkyl (C1-C20); hydroxyl (OH), halogen (F, Cl, Br, I); nitrile (CN);boronic acid (BO₂H₂); carboxylic acid (COOH); carboxylic acid ester(COOK″); primary amide (CONH₂); secondary amide (CONHR″); tertiary amide(CONR″R′″); sulfonamide (SO₂NH₂); N-alkylsulfonamide (SONHR″);whereinR″, R′″ are independently selected from a group consisting of a branchedor linear lower alkyl (C1-C2)); phenyl (C6H5); an R″″ substituted phenylring (R″″C6H4); wherein R″″ is defined above; a carboxylic acid (COOH);a carboxylic acid ester (COOR); wherein R is a branched or linear loweralkyl (C1-C20); and cycloalkyl; wherein R″═R′″═(CH2)n;wherein n=2-10.

In another aspect, W may represents a C-5 modified pyrimidine selectedfrom the group consisting of 5-(N-benzylcarboxyamide)-2′-deoxyuridine(BndU a 5-(N-isobutylcarboxyamide)-2′-deoxyuridine (iBudU), a5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU), a5-(N-naphthylmethylcarboxyamide)-2′-deoxyuridine (NapdU) and acombination thereof.

In another aspect, W represents a C-5 modified pyrimidine selected fromthe group consisting of a 5-(N-tryptaminocarboxyamide)-2′-deoxyuridine(TrpdU) and a 5-(N-naphthylmethylcarboxyamide)-2′-deoxyuridine (NapdU)and a combination thereof.

In another aspect, W may represent a compound comprising the structureshown in Formula I:

whereinR is independently a —(CH₂)_(n)—, wherein n is an integer selected from0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;R^(X1) is independently selected from the group consisting of:

wherein * denotes the point of attachment of the R^(X1) group to the—(CH₂)_(n)— group; andwhereinR^(X4) is independently selected from the group consisting of asubstituted or unsubstituted branched or linear lower alkyl (C1-C20); ahydroxyl group; a halogen (F, Cl, Br, I); nitrile (CN); boronic acid(BO₂H₂); carboxylic acid (COOH); carboxylic acid ester (COOR^(X2));primary amide (CONH₂); secondary amide (CONHR^(X2)); tertiary amide(CONR^(X2)R^(X3)); sulfonamide (SO₂NH₂); N-alkylsulfonamide(SONHR^(X2));R^(X2) and R^(X3) are independently, for each occurrence, selected fromthe group consisting of a substituted or unsubstituted branched orlinear lower alkyl (C1-C20); phenyl (C₆H₅); an R^(X4) substituted phenylring (R^(X4)C₆H₄), wherein R^(X4) is defined above; a carboxylic acid(COOH); a carboxylic acid ester (COOR^(X5)), wherein R^(X5) is abranched or linear lower alkyl (C1-C20); and cycloalkyl, wherein R^(X2)and R^(X3) together form a substituted or unsubstituted 5 or 6 memberedring;X is independently selected from the group consisting of —H, —OH, —OMe,—O-allyl, —F, —OEt, —OPr, —OCH₂CH₂OCH₃, —NH₂ and -azido; R′ isindependently selected from the group consisting of a —H, —OAc;—OBz;—P(NiPr₂)(OCH₂CH₂CN); and —OSiMe₂tBu;R″ is independently selected from the group consisting of a hydrogen,4,4′-dimethoxytrityl (DMT) and triphosphate(—P(O)(OH)—O—P(O)(OH)—O—P(O)(OH)₂) or a salt thereof;Z is independently selected from the group consisting of a —H, asubstituted or unsubstituted branched or linear lower alkyl (C1-C4);and salts thereof;with the following exceptions:when n=4, then R^(X1) cannot be H;when n=3, then R^(X1) cannot be CH₃;when n=0, then R^(X1) cannot be —CH(CH₃)₂; andwhen n=2, and R^(X1) is

and R^(X4) is hydroxyl then R^(X1) cannot be

In related aspect n is an integer selected from 1, 2 or 3.

In related aspect, R^(X1) is selected from the group consisting of:

wherein* denotes the point of attachment of the R^(X1) group to the —(CH₂)_(n)—group; andZ is independently selected from the group consisting of a —H, asubstituted or unsubstituted branched or linear lower alkyl (C1-C4).

In related aspect, R^(X4) is independently selected from the groupconsisting of a branched or linear lower alkyl (C1-C6); —OH; —F andcarboxylic acid (COOH).

In related aspect, X is independently selected from the group consistingof —H, —OH, —OMe and —F.

In related aspect, R′ is selected from the group consisting of a —H,—OAc and —P(NiPr₂)(OCH₂CH₂CN).

In related aspect, R″ is a triphosphate(—P(O)(OH)—O—P(O)(OH)—O—P(O)(OH)₂).

In another aspect, the disclosure provides for a compound comprising thestructure selected from the group consisting of Formulas II (BndC), III(PEdC), IV (PPdC), V (NapdC), VI (2NapdC), VII (NEdC) and VIII (2NEdC):

whereinX is independently selected from the group consisting of —H, —OH, —OMe,—O-allyl, —F, —OEt, —OPr, —OCH₂CH₂OCH₃, —NH₂ and -azido.

In another aspect this disclosure, the aptamer may have a dissociationconstant (K_(d)) for its target of about 10 nM or less. In anotherexemplary embodiment, the aptamer has a dissociation constant (K_(d))for the target protein of about 15 nM or less. In yet another exemplaryembodiment, the aptamer has a dissociation constant (K_(d)) for thetarget protein of about 20 nM or less. In yet another exemplaryembodiment, the aptamer has a dissociation constant (K_(d)) for thetarget protein of about 25 nM or less. In yet another exemplaryembodiment, the aptamer has a dissociation constant (K_(d)) for thetarget protein of about 30 nM or less. In yet another exemplaryembodiment, the aptamer has a dissociation constant (K_(d)) for thetarget protein of about 35 nM or less. In yet another exemplaryembodiment, the aptamer has a dissociation constant (K_(d)) for thetarget protein of about 40 nM or less. In yet another exemplaryembodiment, the aptamer has a dissociation constant (K_(d)) for thetarget protein of about 45 nM or less. In yet another exemplaryembodiment, the aptamer has a dissociation constant (K_(d)) for thetarget protein of about 50 nM or less. In yet another exemplaryembodiment, the aptamer has a dissociation constant (K_(d)) for thetarget protein in a range of about 3-10 nM (or 3, 4, 5, 6, 7, 8, 9 or 10nM0. A suitable dissociation constant can be determined with a bindingassay using a multi-point titration and fitting the equationy=(max−min)(Protein)/(K_(d)+Protein)+min. It is to be understood thatthe determination of dissociation constants is highly dependent upon theconditions under which they are measured and thus these numbers may varysignificantly with respect to factors such as equilibration time, etc.In other embodiments, the aptamer has a K_(d) that is less than or equalto the K_(d) of an aptamer selected from SEQ ID NOS: 1-15.

The motif for the aptamer sequence that binds the ClfA protein isrepresented by SEQ ID NO: 13. This sequence motif may be furthergeneralized to the following sequence:

-   -   AWCWGGWWC(N)_(n)AWCWGGWWWWWAAG (SEQ ID NO:15)

The “W” in the sequence represents a position that may be occupied by aC-5 modified pyrimidine, and “N” represents a position that may beoccupied by any unmodified or modified nucleotide or a spacer-sequenceor linker. Further, n may be a number from 1 to 30 (or 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29 or 30), or from 2 to 20 (or 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19 or 20), or from 5 to 18 (or 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18), or from 10 to 16 (or 10,11, 12, 13, 14, 15 or 16), or N is about 16.

In another aspect, the nucleotide sequence may include up to about 100nucleotides, up to about 95 nucleotides, up to about 90 nucleotides, upto about 85 nucleotides, up to about 80 nucleotides, up to about 75nucleotides, up to about 70 nucleotides, up to about 65 nucleotides, upto about 60 nucleotides, up to about 55 nucleotides, up to about 50nucleotides, up to about 45 nucleotides, up to about 40 nucleotides, upto about 35 nucleotides, up to about 30 nucleotides, up to about 25nucleotides, and up to about 20 nucleotides.

In another aspect this disclosure, the aptamer may be at least about 95%identical, at least about 90% identical, at least about 85% identical,at least about 80% identical, or at least about 75% identical to any ofSEQ ID NO:15. In another embodiment, the aptamer includes a sequencefragments of SEQ ID NO:15.

In another aspect, the aptamer comprises from about 1% to 100% (or 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99 or 100%) C-5 modified pyrimidines. In another aspect, theaptamer comprises from about 10% to about 50% (or 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50%)C-5 modified pyrimidines. In another aspect, the aptamer comprises, fromabout 20% to about 40% (or 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39 or 40%) C-5 modified pyrimidines. Inanother aspect, the aptamer comprises from about 25% to about 35% (or25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35%) C-5 modified pyrimidines.In another aspect, the aptamer comprises from about 27% to about 33% (or27, 28, 29, 30, 31, 32 or 33%) C-5 modified pyrimidines. In anotheraspect, the aptamer comprises from about 37% to about 43% (or 37, 38,39, 40, 41, 42, 43%) C-5 modified pyrimidines.

In another aspect, the W may represent a C-5 modified uridine orcytidine.

In another aspect, the W may represent a C-5 modified pyrimidineillustrated immediately below:

wherein R′ is defined as follows:

whereinR″″ is selected from the group consisting of a branched or linear loweralkyl (C1-C20); hydroxyl (OH), halogen (F, Cl, Br, I); nitrile (CN);boronic acid (BO₂H₂); carboxylic acid (COOH); carboxylic acid ester(COOK″); primary amide (CONH₂); secondary amide (CONHR″); tertiary amide(CONR″R′″); sulfonamide (SO₂NH₂); N-alkylsulfonamide (SONHR″);whereinR″, R′″ are independently selected from a group consisting of a branchedor linear lower alkyl (C1-C2)); phenyl (C6H5); an R″″ substituted phenylring (R″″C6H4); wherein R″″ is defined above; a carboxylic acid (COOH);a carboxylic acid ester (COOR); wherein R is a branched or linear loweralkyl (C1-C20); and cycloalkyl; wherein R″═R′″═(CH2)n;wherein n=2-10.

In another aspect, the W may represents a C-5 modified pyrimidineselected from the group consisting of5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU), a5-(N-isobutylcarboxyamide)-2′-deoxyuridine (iBudU), a5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU), a5-(N-naphthylmethylcarboxyamide)-2′-deoxyuridine (NapdU) and acombination thereof.

In another aspect, the W may represents a C-5 modified pyrimidineselected from the group consisting of a5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU) and a5-(N-naphthylmethylcarboxyamide)-2′-deoxyuridine (NapdU) and acombination thereof. In another aspect, the W may represent a compoundcomprising the structure shown in Formula I:

whereinR is independently a —(CH₂)_(n)—, wherein n is an integer selected from0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;R^(X1) is independently selected from the group consisting of:

wherein * denotes the point of attachment of the R^(X1) group to the—(CH₂)_(n)— group; andwhereinR^(X4) is independently selected from the group consisting of asubstituted or unsubstituted branched or linear lower alkyl (C1-C20); ahydroxyl group; a halogen (F, Cl, Br, I); nitrile (CN); boronic acid(BO₂H₂); carboxylic acid (COOH); carboxylic acid ester (COOR^(X2));primary amide (CONH₂); secondary amide (CONHR^(X2)); tertiary amide(CONR^(X2)R^(X3)); sulfonamide (SO₂NH₂); N-alkylsulfonamide(SONHR^(X2));R^(X2) and R^(X3) are independently, for each occurrence, selected fromthe group consisting of a substituted or unsubstituted branched orlinear lower alkyl (C1-C20); phenyl (C₆H₅); an R^(X4) substituted phenylring (R^(X4)C₆H₄), wherein R^(X4) is defined above; a carboxylic acid(COOH); a carboxylic acid ester (COOR^(X5)), wherein R^(X5) is abranched or linear lower alkyl (C1-C20); and cycloalkyl, wherein R^(X2)and R^(X3) together form a substituted or unsubstituted 5 or 6 memberedring;X is independently selected from the group consisting of —H, —OH, —OMe,—O-allyl, —F, —OEt, —OPr, —OCH₂CH₂OCH₃, —NH₂ and -azido;R′ is independently selected from the group consisting of a —H,—OAc;—OBz; —P(NiPr₂)(OCH₂CH₂CN); and —OSiMe₂tBu;R″ is independently selected from the group consisting of a hydrogen,4,4′-dimethoxytrityl (DMT) and triphosphate(—P(O)(OH)—O—P(O)(OH)—O—P(O)(OH)₂) or a salt thereof;Z is independently selected from the group consisting of a —H, asubstituted or unsubstituted branched or linear lower alkyl (C1-C4);and salts thereof;with the following exceptions:when n=4, then R^(X1) cannot be H;when n=3, then R^(X1) cannot be CH₃;when n=0, then R^(X1) cannot be —CH(CH₃)₂; andwhen n=2, and R^(X1) is

and R^(X4) is hydroxyl then R^(X1) cannot be

In related aspect n is an integer selected from 1, 2 or 3.

In related aspect, R^(X1) is selected from the group consisting of:

wherein* denotes the point of attachment of the R^(X1) group to the —(CH₂)_(n)—group; andZ is independently selected from the group consisting of a —H, asubstituted or unsubstituted branched or linear lower alkyl (C1-C4).In related aspect, R^(X4) is independently selected from the groupconsisting of a branched or linear lower alkyl (C1-C6); —OH; —F andcarboxylic acid (COOH).

In related aspect, X is independently selected from the group consistingof —H, —OH, —OMe and —F.

In related aspect, R′ is selected from the group consisting of a —H,—OAc and —P(NiPr₂)(OCH₂CH₂CN).

In related aspect, R″ is a triphosphate(—P(O)(OH)—O—P(O)(OH)—O—P(O)(OH)₂).

In another aspect, the disclosure provides for a compound comprising thestructure selected from the group consisting of Formulas II (BndC), III(PEdC), IV (PPdC), V (NapdC), VI (2NapdC), VII (NEdC) and VIII (2NEdC):

whereinX is independently selected from the group consisting of —H, —OH, —OMe,—O-allyl, —F, —OEt, —OPr, —OCH₂CH₂OCH₃, —NH₂ and -azido.

In another aspect this disclosure, the aptamer may have a dissociationconstant (K_(d)) for its target of about 10 nM or less. In anotherexemplary embodiment, the aptamer has a dissociation constant (K_(d))for the target protein of about 15 nM or less. In yet another exemplaryembodiment, the aptamer has a dissociation constant (K_(d)) for thetarget protein of about 20 nM or less. In yet another exemplaryembodiment, the aptamer has a dissociation constant (K_(d)) for thetarget protein of about 25 nM or less. In yet another exemplaryembodiment, the aptamer has a dissociation constant (K_(d)) for thetarget protein of about 30 nM or less. In yet another exemplaryembodiment, the aptamer has a dissociation constant (K_(d)) for thetarget protein of about 35 nM or less. In yet another exemplaryembodiment, the aptamer has a dissociation constant (K_(d)) for thetarget protein of about 40 nM or less. In yet another exemplaryembodiment, the aptamer has a dissociation constant (K_(d)) for thetarget protein of about 45 nM or less. In yet another exemplaryembodiment, the aptamer has a dissociation constant (K_(d)) for thetarget protein of about 50 nM or less. In yet another exemplaryembodiment, the aptamer has a dissociation constant (K_(d)) for thetarget protein in a range of about 3-10 nM (or 3, 4, 5, 6, 7, 8, 9 or 10nM0. A suitable dissociation constant can be determined with a bindingassay using a multi-point titration and fitting the equationy=(max−min)(Protein)/(K_(d)+Protein)+min. It is to be understood thatthe determination of dissociation constants is highly dependent upon theconditions under which they are measured and thus these numbers may varysignificantly with respect to factors such as equilibration time, etc.In other embodiments, the aptamer has a K_(d) that is less than or equalto the K_(d) of an aptamer selected from SEQ ID NOS: 1-15.

The present disclosure further provides a method for detecting thepresence or absence of a microorganism in a sample comprising:contacting the sample with an aptamer and performing an assay to detectthe aptamer, wherein detecting the second aptamer indicates that themicroorganism is present in the sample, and wherein not detecting thesecond aptamer indicates that the microorganism is absent from thesample; wherein, the aptamer comprises a nucleic acid molecule havingthe sequence selected from the group consisting ofGGCWWCGGGWACCWAWWAWNGGWWWAGCC(N)_(x)GWC (SEQ ID NO:14) andAWCWGGWWC(N)_(y)WCWGGWWWWWAAG (SEQ ID NO:15), and wherein W isindependently, for each occurrence, a C-5 modified pyrimidine, N is anyunmodified or modified nucleotide, and x is 0, 1, 2, 3, 4 or 5, and y is0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30.

In another aspect, the C-5 modified pyrimidine is selected from thegroup consisting of 5-(N-benzylcarboxyamide)-2′-deoxycytidine (BndC);5-(N-2-phenylethylcarboxyamide)-2′-deoxycytidine (PEdC);5-(N-3-phenylpropylcarboxyamide)-2′-deoxycytidine (PPdC);5-(N-1-naphthylmethylcarboxyamide)-2′-deoxycytidine (NapdC);5-(N-2-naphthylmethylcarboxyamide)-2′-deoxycytidine (2NapdC);5-(N-1-naphthyl-2-ethylcarboxyamide)-2′-deoxycytidine (NEdC);5-(N-2-naphthyl-2-ethylcarboxyamide)-2′-deoxycytidine (2NEdC);5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU);5-(N-isobutylcarboxyamide)-2′-deoxyuridine (iBudU);5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU);5-(N-[1-(3-trimethylammonium) propyl]carboxyamide)-2′-deoxyuridinechloride and 5-(N-naphthylmethylcarboxyamide)-2′-deoxyuridine (NapdU).

In another aspect, the aptamer is amplifiable.

In another aspect, the assay is selected from the group consisting ofPCR, qPCR, mass spectroscopy, sequencing and hybridization.

In another aspect, the microorganism is selected from the groupconsisting of a bacterial cell, parasite and virus. In a related aspect,the microorganism is a bacterial cell. In yet another related aspect,the bacterial cell is pathogenic.

In another aspect, the bacterial cell is a Staphylococcus cell.

In another aspect, the bacterial cell is a Staphylococcus aureus cell.

In another aspect, the aptamer comprises a nucleic acid molecule havinga sequence selected from the group consisting of SEQ ID NOs: 1-8 and10-12, wherein W is a C-5 modified pyrimidine.

In another aspect, the disclosure provides a composition comprising SEQID NOs: 1-15.

The following examples are provided to illustrate certain particularfeatures and/or embodiments. These examples should not be construed tolimit the disclosure to the particular features or embodimentsdescribed.

EXAMPLES Example 1 Selection and Identification of Aptamers HavingBinding Specificity to S. aureus Proteins

This example provides the representative method for the selection andproduction of aptamers having binding specificity to the following tensurface-associated S. aureus proteins: SpA, ClfA, ClfB, FnbA, FnbB,SasD, IsdA, IsdB, IsdC and IsdH.

Purification of S. aureus Targets

Relevant portions of the genes encoding the desired targets or targetdomains were PCR-amplified from S. aureus NRS384 (USA300) genomic DNAwith primers and cloned into pCR-Script SK+ (Stratgene). The clfA, cam,fnbA, sasD, isdA, isdB, isdC, and isdH genes were transferred asBamHI-SacI cassettes into the expression vector pET-51b (EMD-Novagen)that harbors an aminoterminal Strep-tag and a carboxyterminal His₁₀-tag.One of the targets, fnbB, was cloned as and NdeI-BamHI fragment intopET-14b (EMD-Novagen), which harbors an amino-terminal His₁₀-tag. Theplasmids were sequenced to verify the gene identity and proper genefusion of the cloned DNA fragment with the vector-encoded sequences forthe His-tag and Strep tag.

The recombinant proteins were over-expressed in E. coli BL21(DE3) or inBL21(DE3)/pLysE (EMD/Novagen). Conditions for optimal expression ofsoluble proteins were optimized with respect to growth temperature(25-37° C.) and induction time (4-15 h). Cells from 0.1-0.8 l cultureswere lysed with 10 ml BugBuster/Benzonase reagent (EMD Millipore). Therecombinant, His₁₀/Strep-tagged proteins were purified from the solublefraction via sequential affinity chromatography on Ni-NTA agarose andStrep•Tactin® Superflow™ agarose (EMD Millipore). Native staphylococcalprotein A was purchased from VWR and was biotinylated withNHS-PEG4-biotin (Pierce Biotechnology). Protein concentrations weredetermined using the Quick Start Bradford Protein Assay Kit (BioRad).

All 10 recombinant S. aureus cell surface proteins were found in thesoluble fraction when over-expressed in E. coli. Sequential affinitychromatography on Ni-NTA agarose and Streptactin Sepharose yielded0.1-1.5 mg of each protein in >95% purity (see FIG. 3).

Aptamer Selection

Separate libraries with 5-(N-benzylcarboxyamide)-dU (BndU),5-(N-naphthylmethylcarboxyamide)-dU (NapdU), and5-(N-tryptaminocarboxyamide)-dU (TrpdU) were used for SELEX with the S.aureus proteins. Each selection started from 1 nmol (10¹⁴-10¹⁵)sequences containing 40 consecutive randomized positions flanked byfixed sequences required for PCR amplification. SELEX was performedessentially as described (Gold et al., 2010; Vaught et al., 2010;Ochsner et al., 2013). Buffer SB18T was used through-out SELEX andsubsequent binding assays, consisting of 40 mmol 1⁻¹ HEPES pH 7.5, 0.1mol 1⁻¹ NaCl, 5 mmol 1⁻¹ KCl, 5 mmol 1⁻¹ MgCl₂, and 0.05% Tween-20.Eight rounds of selection were carried out, and, beginning with round 2,a kinetic challenge with 10 mmol 1⁻¹ dextran sulfate was performed tofavor slow off-rates. Partitioning of the aptamer-target complexes wasachieved with paramagnetic Talon Dynabeads® Talon® (Invitrogen) thatbind the His₁₀-tag on the recombinant proteins, or with MyOneStreptavidin Cl beads (Life Technologies) for the biotinylated SPA.Selected sequences were eluted from the bead-bound targets with 80 μl 40mmol 1⁻¹ NaOH, neutralized with 20 μl of 160 mmol 1⁻¹ HCl, andPCR-amplified using KOD EX DNA polymerase (Invitrogen-LifeTechnologies). Modified DNA for the next round was prepared with KOD EXDNA polymerase via primer extension from the antisense strand of the PCRproducts and purified as described (Gold et al., 2010).

DNA reassociation kinetic analysis (C₀t) of selected DNA from rounds 3through 8 was used for the assessment of sequence convergence during thelater rounds, indicating increased abundance of some sequences orsequence families. Aptamer pools that demonstrated good affinity(K_(d)≦10 nmol 1⁻¹) in solution binding radioassays (see below) werecloned and the sequences of 48 clones per pool were determined. Up to 12individual aptamers were chosen based on sequence patterns and diversityand prepared enzymatically for further characterization.

Synthetic aptamers were prepared as 48-50-mers at 1 μmol scale viastandard phosphoramidite chemistry and HPLC purified. They contained a5′biotin-dA or 5′fluorescein-biotin-dA, and an inverted dT nucleotide atthe 3′ end (3′idT) for added stability to 3′ to 5′ exonucleases.

Eight rounds of SELEX were performed with these proteins, using threeseparate ssDNA libraries, and C₀t reassociation kinetics indicated areduction of sequence complexity. Pool affinity assays confirmed thesuccessful selection of aptamers for a total of 22 pools obtained withthe ssDNA C-5 modified nucleotides BndU, NapdU, or TrpdU, with poolaffinities in the range of 0.13-8.90 nmol 1⁻¹. Specific binding to S.aureus cells, but no binding to S. epidermidis, S. haemolyticus, S.pyogenes, E. faecalis, E. coli, or P. aeruginosa was observed.

Alignment of sequences determined for 48 clones from each pool showedmulti-copy clones and families that shared common sequence patterns.Representative clones were screened in affinity assays, and the K_(d)'sof the aptamers were in the range of 0.03-2.17 nmol 1⁻¹ (Table 1).

TABLE 1 Aptamer for S. aureus cell surface proteins, with affinity(K_(d)) shown for the original full-length sequences obtained in SELEXAptamer Characterization No. of Protein Clone C-5 K_(d) Nt. C-5 % C-5Target Identifier Mod. (nmol l⁻¹) Length Mods. Mods. SPA 4520-8  NapdU0.22 40 12  30% 4531-56 TrpdU 0.03 39 12 30.8% ClfA 4503-73 BndU 0.79 4015 37.5% 4522-5  TrpdU 0.35 39 12 30.8% ClfB 4504-27 BndU 1.35 40 1947.5% 4511-67 NapdU 3.90 40 16  40% 4523-79 TrpdU 0.47 40 13 32.5% FnbA4726-44 NapdU 4.38 40 8  20% 4745-51 TrpdU 0.63 40 10  25% FnbB 4506-13BndU 4.73 39 21 52.5% 4516-29 NapdU 0.63 40 11 27.5% 4527-83 TrpdU 0.8440 13 32.5% IsdA 4727-62 NapdU 0.73 40 11 27.5% 4746-3  TrpdU 0.16 40 12 30% IsdB 4728-7  NapdU 0.14 40 9 22.5% 4747-90 TrpdU 1.98 40 9 22.5%IsdC 4507-52 BndU 0.15 40 18  45% 4517-71 NapdU 0.08 40 12  30% 4528-22TrpdU 0.07 40 14  35% IsdH 4731-69 NapdU 1.30 40 11 27.5% SasD 4730-3 NapdU 2.17 40 10  25%

The aptamers of Table 1, generally, are from 39 to 40 nucleotides inlength and comprise a C-5 modified pyrimidine (e.g., BndU, TrpdU or aNapdU). Further, the aptamers of able 1 comprise from about 8 to about21 C-5 modified pyrimidines (8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19 or21 C-5 modified pyrimidines), or from about 20% C-5 modified pyrimidinesto about 53% C-5 modified pyrimidines (or from 20%, 22.5%, 25%, 27.5%,30%, 30.8%, 32.5%, 35%. 37.5%, 40%, 45%, 47.5% or 52.5%). The aptamersof table 1, generally, have a K_(d) of from about 0.03 nM to about 4.7nM (or 0.03, 0.07, 0.08, 0.14, 0.15, 0.16, 0.22, 0.35, 0.47, 0.63, 0.73,0.79, 0.84, 1.3, 1.35, 1.98, 2.17, 3.9 and 4.73 nM).

The nucleotide sequence of selected clones that target the SPA proteinand separately the ClfA protein are identified in Table 2 below.

TABLE 2Select Aptamer Nucleotide Sequences of Aptamers Identified via SELEXClone SEQ ID Target Ident. NO: Nucleotide Sequence (5′ to 3′) SPA*4520-3  1         CCGGC WW CGGG W ACC W A WW A W CGG WWW AGCCCAG W CATAA4520-8  2          W CGGC WW CGGG W ACC W A WW A W CGG WWW AGCCCAG WCAGAA 4520-20  3         GCGGC WW CGGG W ACC W A WW A W CGG WWW AGCCCAGW CAAAA 4520-23  4         G W GGC WW CGGG W ACC W A WW A W CGG WWWAGCCCAG W CAGAA 4520-27  5         GCGGC WW CGGG W ACC W A WW A W CGGWWW AGCCC W G W CAGGA 4520-30  6 G W GA W CGAGCGGC WW CGGG W ACC W A WWA WW GG WWW AGCCCAG W CAGAA 4520-42  7          W CGGC WW CGGG W ACC W AWW A W CGG WWW AGCCCAG W CWGAA 4520-44  8         ACGGC WW CGGG W ACC WA WW A W CGG WWW AGCC-AG W CAGAA SPA Seq.  9           GGC WW CGGG W ACCW A WW A W -GG WWW AGCC--G W C Motif ClfA⁺ 4503-66 10 A W CWGG WW CAAAGW GACGA WW GGGCA W C W GG WWWW AAG W 4503-68 11 A W CWGG WW C W AAG WWAC WW GGCG W AA W C W GG WWWW AAGA 4503-73 12 A W CWGG WW CAAAG W GGCGAWW GGGCA W C W GG WWWW AAG W ClfA 13 A W CWGG WW C----------------A W CW GG WWWW AAG Seq. Motif *indicates that the nucleotide ″W″ in thesequences that target the SPA protein are a C-5 modified nucleotide(specifically a NapdU) ⁺indicates that the nucleotide ″W″ in thesequences that target the ClfA protein are a C-5 modified nucleotide(specifically a BndU)

The motif (4520) for the aptamer sequence that binds the SPA protein isrepresented by SEQ ID NO:9. This sequence motif may be furthergeneralized to the following sequence:

(SEQ ID NO: 14) GGCWWCGGGWACCWAWWAWNGGWWWAGCC(N)_(n)GWC.

The “W” in the sequence represents a position that may be occupied by aC-5 modified pyrimidine, and “N” represents a position that may beoccupied by any unmodified or modified nucleotide, and n is from 0 to 2(or 0, 1 or 2).

The motif (4503) for the aptamer sequence that binds the ClfA protein isrepresented by SEQ ID NO:13. This sequence motif may be furthergeneralized to the following sequence:

(SEQ ID NO: 15) AWCWGGWWC(N)_(n)AWCWGGWWWWWAAG

The “W” in the sequence represents a position that may be occupied by aC-5 modified pyrimidine, and “N” represents a position that may beoccupied by any unmodified or modified nucleotide. Further, n may be anumber from 1 to 30 (or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or), or from2 to 20 (or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19 or 20), or from 5 to 18 (or 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17 or 18), or from 10 to 16 (or 10, 11, 12, 13, 14, 15 or 16).

Example 2 Binding and Selective Capture of Bacterial Cells by Aptamers

This example shows that the aptamers selected and identified to bind S.aureus cell surface proteins also bind whole cells and are capable ofselectively capturing S. aureus cells in a mixed bacterial culture

Aptamer Equilibrium and Whole Cell Radiolabel Binding Assays

Aptamers were properly folded via heating for 5 min at 95° C., followedby cooling to room temperature over a 10-15 min period, prior to bindingassays.

Affinities (K_(d)'s) were determined in equilibrium solution bindingassays of radiolabeled aptamers (10-20 pmol 1⁻¹) with serially dilutedproteins (0.001-100 nmol 1⁻¹) and Zorbax PSM-300A (Agilent Technologies)resin for partitioning onto filter plates as described (Gold et al.2010).

Prior to cloning, the aptamer pools were also tested for specificbinding to S. aureus, using S. epidermidis, S. haemolyticus, Strep.pyogenes, Ent. faecalis, E. coli, and Ps. aeruginosa as controls in 2 hequilibrium binding assays. Cell densities ranged from 10⁵-10⁸ CFU mL⁻¹,and 0.1 mmol 1⁻¹ dextran sulfate and 0.35 mol 1⁻¹ NaCl was added to thebinding buffer to reduce non-specific background. In addition,individual aptamers were screened for binding to eight different S.aureus strains belonging to different lineages, including NRS382,NRS383, NRS384, NRS123, NRS385, NRS386, NRS103 (NARSA), and ATCC 29213(ATCC).

Aptamer binding affinities to purified S. aureus proteins correlatedwell with the observed binding to whole bacteria. Two of the SpA-NapdUclones (4520-8 and 4520-9) and three of the SpA-TrpdU clones (4531-55,4531-56, 4531-94) were able to bind whole cells of all S. aureus strainstested, with a detection limit of ˜10⁴ cells per well (10⁵-10⁶ cellsml⁻¹) in a radiolabel filter binding assay. Binding to S. epidermidis orS. haemolyticus cells was not observed, indicating good specificity ofthese aptamers (see FIG. 4B). Similar binding characteristics wereobserved for the ClfA and ClfB aptamers. In contrast, most of the FnbAand FnbB aptamers that strongly bound to S. aureus also had someaffinity to S. epidermidis and S. haemolyticus. aptamers directed to theIsd proteins, in particular IsdC, showed strong and specific binding toS. aureus cells, and signals were enhanced when the bacteria had beengrown under iron-limiting conditions. SasD aptamers failed to bind wholecells, although it is not clear whether this is due to the rather modestaffinity or due to low expression levels of this surface protein

Capture of S. aureus Cells with Cell Surface Protein Directed Aptamers

Biotinylated aptamers were prepared enzymatically via primer extension,using PBDC primers (5′photocleavable biotin, D-spacer and cy3). Forimmobilization, 1 pmol of PBDC aptamers were added to 20 μl MyOneStreptavidin Cl beads (10 mg ml⁻¹), and shaken for 15 min, resulting in˜90% efficiency of immobilization based on cy3 measurements in thenon-captured supernatant fraction. Bacteria were grown for 16 hours at35° C. in LB broth cultures or on tryptic soy agar with 5% sheep bloodand 0.1 mmol 1⁻¹ dipyridyl to create iron-limiting conditions. Cellsuspensions containing up to 10⁶ bacteria in 50 μl SB18T were added tothe capture beads. After incubation with shaking for 1 h at 37° C., thebeads were washed and resuspended in 50 μl SB18T. Cells in thenon-captured supernatant, wash fraction, and on the beads wereenumerated by quantitative plating of serial dilutions onto LB agar.Capture efficiency via quantitative culture was also determined in mixedpopulations and over a range of cell densities (10¹-10⁷ CFU ml⁻¹).

The number of target molecules per cell is unknown for any of thesesurface proteins and expression levels may vary depending on growthconditions and growth phase. However, assuming 1000 copies per cell andusing 10⁷ CFU ml⁻¹ would represent a target concentration of 20 pmol1⁻¹, which is at or below the typical aptamer K_(d)'s. Thus, theradiolabel filter binding assays, where the aptamers are present at lowconcentrations of 10-20 pmol 1⁻¹, is limited to relatively high celldensities. To drive the binding reaction, we used higher concentrations(20 nmol 1⁻¹) of biotinylated SpA aptamers as capture agents attached tobeads, and were able to detect as few as 50 cells in a 0.1 mL sample(FIG. 1A). Aptamer concentrations of 10 nmol 1⁻¹ or above were requiredfor efficient capture of S. aureus at such low cell densities (FIG. 1B).Aptamers were able to bind selectively to S. aureus cells in mixedcultures that contained S. aureus, S. epidermidis, and E. coli each at10⁵-10⁶ CFU ml⁻¹. The best performing binding agents were SpA 4520-8 andClfA 4503-73, demonstrating low non-specific binding comparable torandom sequence modified aptamer controls. Capture of S. aureus onparamagnetic aptamer beads was efficient over a wide range of celldensities, from 5×10² to 5×10⁹ CFU ml⁻¹ (FIG. 5).

Example 3 Enhanced Detection of S. aureus Using Aptamer-Based Enrichment

This example provides exemplary methods for enhancing the detection of amicroorganism (e.g., S. aureus) in a sample by enriching themicroorganism in the sample by aptamer based capture followed by asubsequent detection method (e.g., PCR).

Capture of S. aureus cells was also achieved with 25 nmol 1⁻¹ ofsynthetic, biotinylated aptamers (50 mers) attached to paramagnetic SAbeads (15 min, 37° C., with intermittent shaking) The beads were washedtwice with 100 μl of SB18 to remove any unbound cells, and resuspendedin 50 μl SB18. Full-length, amplifiable aptamers were added (50 μl of 20nmol 1⁻¹), and the beads were incubated for 15 min at 37° C. withintermittent shaking to allow coating of the cells with these surfacecomponent specific aptamers. After washing five times for 2 min eachwith 100 μl of SB18/1 mmol 1⁻¹ dextran sulfate/0.01% Tween-20 and twicewith 100 μl of SB18, bound aptamers were eluted, cleaned up on primercapture beads, and used for qPCR with primers specific for the 5′ and 3′fixed regions as described (Gold et al., 2010).

Capture of S. aureus cells proved useful for downstream detection byPCR, either for enrichment of the sample when cell densities were low,or to remove PCR inhibitors. Coating of the S. aureus cell surface withfull-length, amplifiable aptamers allowed the faster detection by qPCRof the aptamers compared to qPCR of a genomic target, since each cellcontained hundreds of copies of the target surface component fordetection, compared to a single genome. In the example shown in FIG. 2,S. aureus cells were captured with non-amplifiable ClfA aptamers andcoated with amplifiable SpA aptamers or random sequence aptamerscontrols, followed by qPCR using aptamers-specific primers. Separately,the cells were lysed and subjected to qPCR using S. aureus specificgenomic primers, which was clearly less efficient compared to qPCR ofbound aptamers. An shift by up to eight cycles in qPCR detection wasobserved, from 10 cycles for aptamers qPCR to 18 cycles for genomicqPCR, which is consistent with a ratio of several hundred copies(2⁸=256) of surface-bound aptamers to only a single genome. The methodof ClfA aptamer capture and SpA aptamer detection was specific for S.aureus cells, since S. epidermidis cells that do not possess ClfA or SpAdid not result in any aptamer amplification above background. Capture ofbacteria on beads followed by detection with aptamers not only enabledenrichment from low cell density suspensions, but also allowed theefficient removal of PCR inhibitors. Direct genomic PCR failed whencells were in matrices containing excess salt (e.g., 1 mol 1⁻¹ NaCl or0.5 mol 1⁻¹ KCl) or low levels of solvents (e.g., 5% isopropanol),unless the cells were captured first to remove these known PCRinhibitors (Abu Al-Soud and Radstrom, 1998; Schrader et al., 2012).

REFERENCES

-   Abu Al-Soud, W. and Radstrom, P. (1998) Capacity of nine    thermostable DNA polymerases to mediate DNA amplification in the    presence of PCR-inhibiting samples. Appl Environ Microbiol 64,    3748-3753.-   Cao, X., Li, S., Chen, L., Ding, H., Xu, H., Huang, Y., Li, J.,    Liu, N. et al. (2009) Combining use of a panel of ssDNA aptamers in    the detection of Staphylococcus aureus. Nucleic Acids Res 37,    4621-4628.-   Dreisbach, A., van Dijl, J. M. and Buist, G. (2011) The cell surface    proteome of Staphylococcus aureus. Proteomics 11, 3154-3168.-   Dwivedi, H. P., Smiley, R. D. and Jaykus, L. A. (2013) Selection of    DNA aptamers for capture and detection of Salmonella typhimurium    using a whole-cell SELEX approach in conjunction with cell sorting.    Appl Microbiol Biotechnol 97, 3677-3686.-   Falugi, F., Kim, H. K., Missiakas, D. M. and Schneewind, O. (2013)    Role of protein A in the evasion of host adaptive immune responses    by Staphylococcus aureus. MBio 4, e00575-00513.-   Gill, S. R., Fouts, D. E., Archer, G. L., Mongodin, E. F., Deboy, R.    T., Ravel, J., Paulsen, I. T., Kolonay, J. F. et al. (2005) Insights    on evolution of virulence and resistance from the complete genome    analysis of an early methicillin-resistant Staphylococcus aureus    strain and a biofilm-producing methicillin-resistant Staphylococcus    epidermidis strain. J Bacteriol 187, 2426-2438.-   Gold, L., Ayers, D., Bertino, J., Bock, C., Bock, A., Brody, E. N.,    Carter, J., Dalby, A. B. et al. (2010) Aptamer-based multiplexed    proteomic technology for biomarker discovery. PLoS One 5, e15004.-   Gold, L., Walker, J. J., Wilcox, S. K. and Williams, S. (2012)    Advances in human proteomics at high scale with the SOMAscan    proteomics platform. N Biotechnol 29, 543-549.-   Grigg, J. C., Ukpabi, G., Gaudin, C. F. and Murphy, M. E. (2010)    Structural biology of heme binding in the Staphylococcus aureus Isd    system. J Inorg Biochem 104, 341-348.-   Hussain, M., Becker, K., von Eiff, C., Schrenzel, J., Peters, G. and    Herrmann, M. (2001) Identification and characterization of a novel    38.5-kilodalton cell surface protein of Staphylococcus aureus with    extended-spectrum binding activity for extracellular matrix and    plasma proteins. J Bacteriol 183, 6778-6786.-   Kobayashi, S. D., and Deleo, F. R. (2013) Staphylococcus aureus    Protein A Promotes Immune Suppression. MBio 4, e00764-13.    doi:10.1128-   Marraffini, L. A., Dedent, A. C. and Schneewind, O. (2006) Sortases    and the art of anchoring proteins to the envelopes of gram-positive    bacteria. Microbiol Mol Biol Rev 70, 192-221.-   Mazmanian, S. K., Skaar, E. P., Gaspar, A. H., Humayun, M.,    Gornicki, P., Jelenska, J.,-   Joachmiak, A., Missiakas, D. M. and Schneewind, O. (2003) Passage of    heme-iron across the envelope of Staphylococcus aureus. Science 299,    906-909.-   McCarthy, A. J. and Lindsay, J. A. (2010) Genetic variation in    Staphylococcus aureus surface and immune evasion genes is lineage    associated: implications for vaccine design and host-pathogen    interactions. BMC Microbiol 10, 173.-   McDevitt, D., Nanavaty, T., House-Pompeo, K., Bell, E., Turner, N.,    McIntire, L., Foster, T.-   and Hook, M. (1997) Characterization of the interaction between the    Staphylococcus aureus clumping factor (ClfA) and fibrinogen. Eur J    Biochem 247, 416-424.-   Ni Eidhin, D., Perkins, S., Francois, P., Vaudaux, P., Hook, M. and    Foster, T. J. (1998) Clumping factor B (ClfB), a new surface-located    fibrinogen-binding adhesin of Staphylococcus aureus. Mol Microbiol    30, 245-257.-   Ochsner, U. A., Katilius, E. and Janjic, N. (2013) Detection of    Clostridium difficile toxins A, B and binary toxin with slow    off-rate modified aptamers. Diagn Microbiol Infect Dis. 76, 278-285.-   Palma, M., Wade, D., Flock, M. and Flock, J. I. (1998) Multiple    binding sites in the interaction between an extracellular    fibrinogen-binding protein from Staphylococcus aureus and    fibrinogen. J Biol Chem 273, 13177-13181.-   Rao, S. S., Mohan, K. V., Gao, Y. and Atreya, C. D. (2013)    Identification and evaluation of a novel peptide binding to the cell    surface of Staphylococcus aureus. Microbiol Res 168, 106-112.-   Roche, F. M., Massey, R., Peacock, S. J., Day, N. P., Visai, L.,    Speziale, P., Lam, A., Pallen, M. and Foster, T. J. (2003)    Characterization of novel LPXTG-containing proteins of    Staphylococcus aureus identified from genome sequences. Microbiology    149, 643-654.-   Roche, F. M., Downer, R., Keane, F., Speziale, P., Park, P. W. and    Foster, T. J. (2004) The N-terminal A domain of fibronectin-binding    proteins A and B promotes adhesion of Staphylococcus aureus to    elastin. J Biol Chem 279, 38433-38440.-   Schneewind, O., Fowler, A. and Faull, K. F. (1995) Structure of the    cell wall anchor of surface proteins in Staphylococcus aureus.    Science 268, 103-106.-   Schrader, C., Schielke, A., Ellerbroek, L. and Johne, R. (2012) PCR    inhibitors—occurrence, properties and removal. J Appl Microbiol 113,    1014-1026.-   Sorum, M., Sangvik, M., Stegger, M., Olsen, R. S., Johannessen, M.,    Skov, R. and Sollid, J. U. (2013) Staphylococcus aureus mutants    lacking cell wall-bound protein A found in isolates from    bacteraemia, MRSA infection and a healthy nasal carrier. Pathog Dis    67, 19-24. Speziale, P., Pietrocola, G., Rindi, S., Provenzano, M.,    Provenza, G., Di Poto, A., Visai, L. and Arciola, C. R. (2009)    Structural and functional role of Staphylococcus aureus surface    components recognizing adhesive matrix molecules of the host. Future    Microbiol 4, 1337-1352.-   Stranger-Jones, Y. K., Bae, T. and Schneewind, O. (2006) Vaccine    assembly from surface proteins of Staphylococcus aureus. Proc Natl    Acad Sci USA 103, 16942-16947.-   Timofeyeva, Y., Scully, I. L. and Anderson, A. S. (2014)    Immunofluorescence microscopy for the detection of surface antigens    in methicillin-resistant Staphylococcus aureus (MRSA). Methods Mol    Biol 1085, 85-95.-   Vaught, J. D., Bock, C., Carter, J., Fitzwater, T., Otis, M.,    Schneider, D., Rolando, J., Waugh, S. et al. (2010) Expanding the    chemistry of DNA for in vitro selection. J Am Chem Soc 132,    4141-4151.-   Ythier, M., Resch, G., Waridel, P., Panchaud, A., Gfeller, A.,    Majcherczyk, P., Quadroni, M.,-   and Moreillon, P. (2012) Proteomic and transcriptomic profiling of    Staphylococcus aureus surface LPXTG-proteins: correlation with agr    genotypes and adherence phenotypes. Mol Cell Proteomics 11,    1123-1139.

1-54. (canceled)
 55. A nucleic acid molecule comprising the sequence ofGGCWWCGGGWACCWAWWAWNGGWWWAGCC(N)_(n)GWC (SEQ ID NO:14), wherein W isindependently, for each occurrence, a C-5 modified pyrimidine, N is anyunmodified or modified nucleotide, and n is 0, 1, 2, 3, 4 or
 5. 56. Thenucleic acid molecule of claim 55, wherein the nucleic acid molecule isat least about 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49 or 50 nucleotides in length.
 57. The nucleic acidmolecule of claim 55, wherein the nucleic acid molecule is at from about32 to about 100 nucleotides in length.
 58. A nucleic acid moleculecomprising the sequence of AWCWGGWWC(N)_(n)AWCWGGWWWWWAAG (SEQ IDNO:15), wherein W is independently, for each occurrence, a C-5 modifiedpyrimidine, N is any unmodified or modified nucleotide, and n is 0, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29 or
 30. 59. The nucleic acid molecule ofclaim 58, wherein the nucleic acid molecule is at least about 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides inlength.
 60. The nucleic acid molecule of claim 58, wherein the nucleicacid molecule is from about 18 to about 100 nucleotides in length. 61.The nucleic acid molecule of claim 55, wherein the nucleic acid moleculecomprises a nucleic acid molecule having a sequence selected from thegroup consisting of SEQ ID NOs:1-8, wherein W is a C-5 modifiedpyrimidine.
 62. The nucleic acid molecule of claim 55, wherein the C-5modified pyrimidine is selected from the group consisting of5-(N-benzylcarboxyamide)-2′-deoxycytidine (BndC);5-(N-2-phenylethylcarboxyamide)-2′-deoxycytidine (PEdC);5-(N-3-phenylpropylcarboxyamide)-2′-deoxycytidine (PPdC);5-(N-1-naphthylmethylcarboxyamide)-2′-deoxycytidine (NapdC);5-(N-2-naphthylmethylcarboxyamide)-2′-deoxycytidine (2NapdC);5-(N-1-naphthyl-2-ethylcarboxyamide)-2′-deoxycytidine (NEdC);5-(N-2-naphthyl-2-ethylcarboxyamide)-2′-deoxycytidine (2NEdC);5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU);5-(N-isobutylcarboxyamide)-2′-deoxyuridine (iBudU);5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU);5-(N-[1-(3-trimethylammonium) propyl]carboxyamide)-2′-deoxyuridinechloride and 5-(N-naphthylmethylcarboxyamide)-2′-deoxyuridine (NapdU).63. (canceled)
 64. The nucleic acid molecule of claim 55, wherein C-5modified pyrimidine is a5-(N-naphthylmethylcarboxyamide)-2′-deoxyuridine (NapdU).
 65. Thenucleic acid molecule of claim 61, wherein the C-5 modified pyrimidineis selected from the group consisting of5-(N-benzylcarboxyamide)-2′-deoxycytidine (BndC);5-(N-2-phenylethylcarboxyamide)-2′-deoxycytidine (PEdC);5-(N-3-phenylpropylcarboxyamide)-2′-deoxycytidine (PPdC);5-(N-1-naphthylmethylcarboxyamide)-2′-deoxycytidine (NapdC);5-(N-2-naphthylmethylcarboxyamide)-2′-deoxycytidine (2NapdC);5-(N-1-naphthyl-2-ethylcarboxyamide)-2′-deoxycytidine (NEdC);5-(N-2-naphthyl-2-ethylcarboxyamide)-2′-deoxycytidine (2NEdC);5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU);5-(N-isobutylcarboxyamide)-2′-deoxyuridine (iBudU);5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU);5-(N-[1-(3-trimethylammonium) propyl]carboxyamide)-2′-deoxyuridinechloride and 5-(N-naphthylmethylcarboxyamide)-2′-deoxyuridine (NapdU).66. The nucleic acid molecule of claim 61, wherein C-5 modifiedpyrimidine is a 5-(N-naphthylmethylcarboxyamide)-2′-deoxyuridine(NapdU).
 67. The nucleic acid molecule of claim 58, wherein the nucleicacid molecule comprises a nucleic acid molecule having a sequenceselected from the group consisting of SEQ ID NOs:10-12, wherein W is aC-5 modified pyrimidine.
 68. The nucleic acid molecule of claim 58,wherein the C-5 modified pyrimidine is selected from the groupconsisting of 5-(N-benzylcarboxyamide)-2′-deoxycytidine (BndC);5-(N-2-phenylethylcarboxyamide)-2′-deoxycytidine (PEdC);5-(N-3-phenylpropylcarboxyamide)-2′-deoxycytidine (PPdC);5-(N-1-naphthylmethylcarboxyamide)-2′-deoxycytidine (NapdC);5-(N-2-naphthylmethylcarboxyamide)-2′-deoxycytidine (2NapdC);5-(N-1-naphthyl-2-ethylcarboxyamide)-2′-deoxycytidine (NEdC);5-(N-2-naphthyl-2-ethylcarboxyamide)-2′-deoxycytidine (2NEdC);5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU);5-(N-isobutylcarboxyamide)-2′-deoxyuridine (iBudU);5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU);5-(N-[1-(3-trimethylammonium) propyl]carboxyamide)-2′-deoxyuridinechloride and 5-(N-naphthylmethylcarboxyamide)-2′-deoxyuridine (NapdU).69. The nucleic acid molecule of claim 58, wherein C-5 modifiedpyrimidine is a 5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU).
 70. Thenucleic acid molecule of claim 67, wherein the C-5 modified pyrimidineis selected from the group consisting of5-(N-benzylcarboxyamide)-2′-deoxycytidine (BndC);5-(N-2-phenylethylcarboxyamide)-2′-deoxycytidine (PEdC);5-(N-3-phenylpropylcarboxyamide)-2′-deoxycytidine (PPdC);5-(N-1-naphthylmethylcarboxyamide)-2′-deoxycytidine (NapdC);5-(N-2-naphthylmethylcarboxyamide)-2′-deoxycytidine (2NapdC);5-(N-1-naphthyl-2-ethylcarboxyamide)-2′-deoxycytidine (NEdC);5-(N-2-naphthyl-2-ethylcarboxyamide)-2′-deoxycytidine (2NEdC);5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU);5-(N-isobutylcarboxyamide)-2′-deoxyuridine (iBudU);5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU);5-(N-[1-(3-trimethylammonium) propyl]carboxyamide)-2′-deoxyuridinechloride and 5-(N-naphthylmethylcarboxyamide)-2′-deoxyuridine (NapdU).71. The nucleic acid molecule of claim 67, wherein C-5 modifiedpyrimidine is a 5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU).